RU2793783C1 - Methods and compositions including purified recombinant polypeptides - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: process for purifying a recombinant polypeptide produced in Chinese hamster ovary host cells characterized by levels of phospholipase B-like enzyme 2 (PLBL2) above 100 ng/mg, including a hydrophobic interaction chromatography (HIC) stage is described, where the method provides a purified preparation including a recombinant polypeptide and residual hamster PLBL2.

EFFECT: invention expands the arsenal of methods for purifying recombinant polypeptides.

29 cl, 7 dwg, 16 tbl, 3 ex

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority over US provisional application number 61/877,517, filed September 13, 2013, which is incorporated herein by reference in its entirety.

SEQUENCE LIST

This application contains the sequence listing filed via EFS-Web and incorporated herein by reference in its entirety. The specified ASCII copy was created on August 28, 2014, is called 2014.AUG.28 P5704R1-WO Sequence Listing.txt and has a size of 34,811 bytes.

FIELD OF TECHNOLOGY

Provided are purified recombinant polypeptides isolated from Chinese hamster ovary host cells, including antibodies, such as therapeutic antibodies, and methods for making and using such polypeptides.

BACKGROUND OF THE INVENTION

A number of medicines for the treatment of asthma and other respiratory disorders are commercially available or under development. One of the targets in asthma therapy is IL-13. IL-13 is a TH2 pleiotropic cytokine produced by activated T cells, NKT cells, basophils, eosinophils, and mast cells and has been shown in preclinical models to play an important role in the pathogenesis of asthma. IL-13 antagonists have previously been described, including anti-IL-13 antibodies. Based on some of these antibodies, therapeutic agents have been developed for human use. Recently, a number of studies have demonstrated the clinical activity of anti-IL-13 monoclonal antibodies in the treatment of asthma (See e.g. Corren et al., 2011, N. Engl. J. Med. 365, 1088-1098; Gauvreau et al., 2011 , Am. J. Respir. Crit. Care Med. 183, 1007-1014; Ingram and Kraft, 2012, J. Allergy Clin. Immunol. 130, 829-42; Webb, 2011, Nat Biotechnol 29, 860-863). Among them, lebrikizumab, a humanized IgG4 antibody that neutralizes IL-13 activity and improves lung function in asthmatics, who, despite treatment, which, in most cases, was carried out with inhaled corticosteroids and a long-acting beta2-adrenergic receptor agonist, experienced symptoms of the disease ( Corren et al., 2011, N. Engl. J. Med. 365, 1088-1098).

In addition, IL-13 has been shown to be involved in many other allergic and fibrotic disorders. For example, such IL13-mediated diseases and/or conditions include allergic asthma, non-allergic (endogenous) asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergies, chronic obstructive pulmonary disease, ulcerative colitis, RSV infections, uveitis, scleroderma. and osteoporosis, but are not limited to.

In order for recombinant biopharmaceutical proteins to be approved for administration to human patients, it is important to remove residual impurities from the final biological product, which are formed during their production and purification. These components associated with the production process include culture medium proteins, immunoglobulin affinity ligands, viruses, endotoxin, DNA, and host cell proteins. These host cell contaminants include process-specific host cell proteins (HCPs), which are process-related contaminants/contaminants in biologicals produced by recombinant DNA technology. Although HCPs are generally present in the finished drug substance in small amounts (parts per million or nanograms per milligram of the intended recombinant protein), it should be recognized that HCPs are undesirable and their amount should be minimized. For example, the US Food and Drug Administration (FDA) requires biopharmaceuticals intended for in vivo human use to contain as few contaminants as possible, and also requires testing to detect and quantify potential contaminants/impurities, such as HCP.

Methods for purifying proteins from cellular debris initially depend on the site of protein expression. Some proteins are secreted by cells directly into the environment for growth; others are produced intracellularly. For the latter proteins, the first step in the purification process involves cell lysis, which can be carried out in a variety of ways, including mechanical action, osmotic shock, or enzymatic treatment. As a result of such destruction, the entire content of the cells is released into the homogenate, but subcellular fragments are formed, which are difficult to remove due to their small size. They are usually removed by centrifugation or filtration. The same problem arises in the case of secreted proteins, as natural cell death occurs and intracellular proteins are released from host cells during the protein production cycle.

Once a solution containing a protein of interest has been obtained, its separation from other proteins produced by the cell is usually achieved using a combination of different chromatographic techniques. As a rule, in these methods, the separation of protein mixtures is carried out by charge, degree of hydrophobicity, or size. A range of different chromatographic resins are available for each of these methods, allowing fine-tuning of the purification scheme for a particular protein. The essence of all these methods of separation is that proteins can either move at different speeds down a long column, due to which their physical separation is achieved, which is the greater the farther the proteins go down the column, or proteins can selectively bind to the separation medium and further differentially eluted with different solvents. In some cases, the target protein is separated from the impurities when the impurities specifically bind to the column and the target protein does not, i.e. the target protein remains in the "breakthrough".

Ion exchange chromatography is a procedure applicable to the purification of ionized molecules. Ionized molecules are separated based on the non-specific electrostatic interaction of their charged groups with oppositely charged molecules attached to the solid phase substrate, as a result, those ionized molecules that interact more strongly with the solid phase are retained. The net charge of each type of ionized molecule and its affinity for the matrix varies depending on the number of charged groups, the charge of each group, and the nature of the molecules competing for interaction with the charged solid state matrix. Because of these differences, different types of molecules are separated using ion exchange chromatography. In a typical protein purification procedure using ion exchange chromatography, a mixture of a plurality of proteins derived from a host cell, such as in mammalian cell culture, is applied to an ion exchange column. After non-binding molecules pass through the column (wash out), the conditions are adjusted, for example, by changing the pH, the concentration of counterions, and the like, in a stepwise or gradient mode, in order to remove non-specifically retained ionized target protein from the solid phase, separating it from proteins with different charge characteristics. Anion exchange chromatography involves competition between a target anionic molecule and a negatively charged counterion for interaction with a positively charged molecule attached to a solid phase matrix, at pH values and under the conditions of a particular separation process. In contrast, cation exchange chromatography involves competition between a target cationic molecule and a positively charged counterion for interaction with a negatively charged molecule attached to a solid phase matrix at pH values and under the conditions of a particular separation process. Combined ion exchange chromatography (also called multimodal ion exchange chromatography) involves the use of a combination of cation exchange and anion exchange chromatography media in one step. In particular, "combined" refers to a solid phase support to which a mixture of cation exchange, anion exchange, and hydrophobic moieties is covalently attached.

Chromatography of proteins on a hydroxyapatite column involves the non-specific interaction of charged amino groups or carboxyl groups of a protein with oppositely charged groups on hydroxyapatite, where the net charge of hydroxyapatite and protein is controlled by the pH of the buffer. The elution is carried out by replacing the non-specific protein-hydroxyapatite pair with ions such as Ca ²⁺ or Mg ²⁺ . The negatively charged groups of the protein are replaced by negatively charged compounds such as phosphates, resulting in the elution of the protein having a negative net charge.

Hydrophobic interaction chromatography (HIC) is typically used to purify and separate molecules such as proteins based on differences in the hydrophobicity of their surfaces. The hydrophobic groups of the protein interact non-specifically with the hydrophobic groups associated with the chromatographic matrix. Differences in the number and nature of hydrophobic groups on the surface of the protein cause proteins to move at different speeds along the HIC column (unequal retention of proteins on the HIC column), which leads to the separation of proteins in complex protein mixtures.

Affinity chromatography, based on a specific, structure-dependent (ie, spatial complementarity) interaction between the protein to be purified and the immobilized capture agent, is a standard option for the purification of some proteins, such as antibodies. Protein A, for example, is used as an adsorbent for the affinity chromatography of proteins such as antibodies containing an Fc region. Protein A is a 41 kDa Staphylococcus aureas cell wall protein that binds with high affinity (about 10 ^-8 M for human IgG) to the Fc region of antibodies.

Purification of recombinant polypeptides is typically carried out using binding and elution chromatography (B/E) or collection chromatography (F/T). They are briefly described below.

Binding and Elution Chromatography (B/E): During B/E chromatography, the product is typically applied to maximize the dynamic binding capacity (DBC) of the chromatographic material, and the wash and elution conditions are then adjusted to maximize the purity of the product in the eluate.

Various B/E methods have been described for use in conjunction with protein A affinity chromatography, including various intermediate wash buffers. For example, US Pat. bound to protein A on the column. US Pat. No. 6,870,034 describes additional methods and wash buffers for use in the Protein A affinity chromatography process.

Breakthrough Collection Chromatography (F/T): When using F/T chromatography, the application conditions are chosen so that the impurities are strongly bound to the chromatographic material and the product remains in the breakthrough. F/T chromatography provides high coverage for standard monoclonal antibody (MAb) preparations.

We found that in recombinant anti-IL13 MAb preparations and in several other recombinant CHO cell-derived polypeptides, phospholipase B-like enzyme 2 is the only type of CHOP that is present in excess in available antibodies as measured by total CHOP ELISA. As used herein, the terms "PLB2" and "PLBL2" and "PLBD2" are used interchangeably and refer to "phospholipase B-like enzyme 2" or its synonym, "phospholipase B-domain-like enzyme 2". Some scientific publications regarding PLBL2 include Lakomek, K. et al., BMC Structural Biology 9:56 (2009); Deuschi, et al., FEBS Lett 580:5747-5752 (2006). PLBL2 is synthesized as a pre-pro-enzyme with an initial molecular weight of approximately 66,000. The original leader sequence is removed and 6 mannose-6-phosphate (M-6-P) groups are potentially added during post-translational modification. M-6-P is a specific modification that directs this enzyme to the lysosome via the M-6-P receptor. PLBL2 contains 6 cysteines, two of which have free sulfhydryl groups and four form disulfide bonds. Under acidic conditions, PLBL2 is further cleaved into N- and C-terminal fragments having a molecular weight of 32,000 and 45,000, respectively. By analogy with other lysosomal enzymes, this cleavage is an activation step that provides substrate access to the active site.

The homology between the amino acid sequences of hamster and human PLBL2 is approximately 80%. It is believed that the enzyme breaks down any fatty acid chain in the composition of phospholipids that form cell membranes. There are other phospholipases with different substrate specificities. Microorganisms have a similar enzymatic activity, where it is often a virulence factor. Although microorganisms have similar enzymatic activity, the protein providing such activity is different from the mammalian protein, and the homology between the sequences of the PLBL2 enzyme of microorganisms and mammals is low. Phospholipases produce free fatty acids (FFA) as one of the products of substrate hydrolysis. Free fatty acids themselves are a potential immunosignaling factor. As a result of dehydrogenation, FFAs are converted to arachidonic acid, which is potentially involved in inflammatory cascades involving eicosanoids.

Having identified PLBL2 as the only HCP (CHOP) in preparations of recombinant anti-IL13 monoclonal antibodies and several other recombinant polypeptides produced in CHO cells, we have developed reagents, methods and kits for the specific, sensitive and quantitative determination of the level of PLBL2 in preparations of anti-IL- 13 monoclonal antibodies (and other recombinant polypeptide products) at various stages of purification. They are briefly described in the Examples below and also in US Provisional Applications 61/877,503 and 61/991,228. In addition, it has proven challenging to develop a large-scale, reliable, and efficient process for purifying anti-IL13 MAbs (and other recombinant polypeptide products) resulting in sufficiently pure MAbs (including removal of PLBL2) for human therapeutic applications, including late stage applications. clinical and commercial use. The invention described herein satisfies some of the needs described above and provides a number of other advantages.

All references cited in this document, including patent applications and published patent applications, are incorporated herein by reference in their entirety.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the development of an improved process for purifying recombinant polypeptides produced by Chinese Hamster Ovary (CHO) cells, which provides a significant reduction in the level of hamster PLBL2 in the purified product. Recombinant polypeptides purified according to the methods of the present invention, including therapeutic antibodies such as an anti-IL13 antibody, may have reduced immunogenicity when administered to human subjects.

Accordingly, in one aspect, compositions are provided comprising an anti-IL13 monoclonal antibody purified from CHO cells, comprising an anti-IL13 antibody and a residual amount of hamster PLBL2. In some embodiments, the amount of hamster PLBL2 is less than 20 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 15 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 10 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 8 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 5 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 3 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 2 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 1 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 0.5 ng/mg. In some embodiments, the amount of hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg. mg, or between 0.5 ng/mg and 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3 ng/mg, or between 0.5 ng /mg and 2 ng/mg, or between 0.5 ng/mg and 1 ng/mg, or between the limit of quantification in the assay (LOQ) and 1 ng/mg. In some embodiments, the anti-IL13 antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.: 1, CDR-H2 having the amino acid sequence of SEQ ID NO.: 2, and CDR-H3 having the amino acid sequence sequence of SEQ ID NO.: 3, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.: 4, CDR-L2 having the amino acid sequence of SEQ ID NO.: 5, and CDR-L3 having the amino acid sequence SEQ ID NO.: 6. In some embodiments, the anti-IL13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 7. In some embodiments, the anti-IL13 antibody comprises a light chain variable region having the amino acid sequence SEQ ID NO.: 9. In some embodiments, the anti-IL13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO.: 10. In some embodiments, the anti-IL13 antibody comprises a light chain having the amino acid sequence of SEQ ID NO. : 14. In some embodiments, the anti-IL13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO.: 9. In some embodiments, the anti -IL13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO.: 10 and a light chain having the amino acid sequence of SEQ ID NO.: 14. In some embodiments, the amount of hamster PLBL2 in the composition is quantified using immunoassay or mass spectrometric analysis. In some embodiments, the immunoassay is a Chinese Hamster Ovary all protein ELISA or a hamster PLBL2 ELISA. In some embodiments, the mass spectrometry analysis is LC-MS/MS.

In another aspect, anti-IL13 monoclonal antibody preparations are provided that are isolated and purified from CHO cells by a process including a hydrophobic interaction chromatography (HIC) step. In some embodiments, the purified preparation comprises an anti-IL13 antibody and a residual amount of hamster PLBL2. In some embodiments, the amount of hamster PLBL2 is less than 20 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 15 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 10 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 8 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 5 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 3 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 2 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 1 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 0.5 ng/mg. In some embodiments, the amount of hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg. mg, or between 0.5 ng/mg and 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3 ng/mg, or between 0.5 ng /mg and 2 ng/mg, or between 0.5 ng/mg and 1 ng/mg, or between the limit of quantification in the assay (LOQ) and 1 ng/mg. In some embodiments, the HIC step includes the use of PHENYL SEPHAROSE ^TM 6 Fast Flow (High Sub) resin. In some embodiments, the HIC step includes using a resin column in breakthrough collection mode. In some embodiments, the HIC step includes the use of an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer comprise 50 mM sodium acetate, pH 5.0. In some embodiments, breakthrough is monitored by absorbance at 280 nanometers and breakthrough is collected between 0.5 OD and 1.5 OD. In some embodiments, the breakthrough is collected up to a maximum of 8 column volumes. In some embodiments, the process further includes an affinity chromatography step. In some embodiments, the affinity chromatography is protein A chromatography. In some embodiments, the process further includes an ion exchange chromatography step. In some embodiments, the ion exchange chromatography is an anion exchange chromatography. In some embodiments, the anti-IL13 antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.: 1, CDR-H2 having the amino acid sequence of SEQ ID NO.: 2, and CDR-H3 having the amino acid sequence sequence of SEQ ID NO.: 3, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.: 4, CDR-L2 having the amino acid sequence of SEQ ID NO.: 5, and CDR-L3 having the amino acid sequence SEQ ID NO.: 6. In some embodiments, the anti-IL13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 7. In some embodiments, the anti-IL13 antibody comprises a light chain variable region having the amino acid sequence SEQ ID NO.: 9. In some embodiments, the anti-IL13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO.: 10. In some embodiments, the anti-IL13 antibody comprises a light chain having the amino acid sequence of SEQ ID NO. : 14. In some embodiments, the anti-IL13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO.: 9. In some embodiments, the anti -IL13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO.: 10 and a light chain having the amino acid sequence of SEQ ID NO.: 14. In some embodiments, the amount of hamster PLBL2 is quantified using immunoassay or mass spectrometric analysis. . In some embodiments, the immunoassay is a Chinese Hamster Ovary all protein ELISA or a hamster PLBL2 ELISA. In some embodiments, the mass spectrometry analysis is LC-MS/MS.

In yet another aspect, purified anti-IL13 monoclonal antibody preparations isolated from CHO cells are provided. In some embodiments, the antibody preparation is purified by a process comprising a first step of protein A affinity chromatography, a second step of anion exchange chromatography, and a third step of hydrophobic interaction chromatography (HIC), resulting in a purified preparation. In some embodiments, the purified preparation comprises an anti-IL13 antibody and a residual amount of hamster PLBL2. In some embodiments, the amount of hamster PLBL2 is less than 20 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 15 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 10 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 8 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 5 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 3 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 2 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 1 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 0.5 ng/mg. In some embodiments, the amount of hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg. mg, or between 0.5 ng/mg and 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3 ng/mg, or between 0.5 ng /mg and 2 ng/mg, or between 0.5 ng/mg and 1 ng/mg, or between the limit of quantification in the assay (LOQ) and 1 ng/mg. In some embodiments, the affinity chromatography step includes the use of MABSELECT SURE ^TM resin, the anion exchange chromatography step includes the use of Q SEPHAROSE ^TM Fast Flow resin, and the HIC step includes the use of PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) resin. In some embodiments, the affinity chromatography step includes the use of a MABSELECT SURE ^™ resin column in binding and elution mode, the anion exchange chromatography step includes the use of a Q SEPHAROSE ^™ Fast Flow resin column in the binding and elution mode, and the HIC step includes the use of a PHENYL resin column SEPHAROSE ^TM 6 Fast Flow (High Sub) in breakthrough mode. In some embodiments, the anti-IL13 antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.: 1, CDR-H2 having the amino acid sequence of SEQ ID NO.: 2, and CDR-H3 having the amino acid sequence sequence of SEQ ID NO.: 3, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.: 4, CDR-L2 having the amino acid sequence of SEQ ID NO.: 5, and CDR-L3 having the amino acid sequence SEQ ID NO.: 6. In some embodiments, the anti-IL13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 7. In some embodiments, the anti-IL13 antibody comprises a light chain variable region having the amino acid sequence SEQ ID NO.: 9. In some embodiments, the anti-IL13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO.: 10. In some embodiments, the anti-IL13 antibody comprises a light chain having the amino acid sequence of SEQ ID NO. : 14. In some embodiments, the anti-IL13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO.: 9. In some embodiments, the anti -IL13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO.: 10 and a light chain having the amino acid sequence of SEQ ID NO.: 14. In some embodiments, the amount of hamster PLBL2 is quantified using immunoassay or mass spectrometric analysis. . In some embodiments, the immunoassay is a Chinese Hamster Ovary all protein ELISA or a hamster PLBL2 ELISA. In some embodiments, the mass spectrometry analysis is LC-MS/MS.

In yet another aspect, methods are provided for purifying a recombinant protein produced in CHO cells, wherein the method provides a purified preparation comprising the recombinant polypeptide and a residual amount of hamster PLBL2. In some embodiments, the amount of hamster PLBL2 is less than 20 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 15 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 10 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 8 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 5 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 3 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 2 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 1 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 0.5 ng/mg. In some embodiments, the amount of hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg. mg, or between 0.5 ng/mg and 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3 ng/mg, or between 0.5 ng /mg and 2 ng/mg, or between 0.5 ng/mg and 1 ng/mg, or between the limit of quantification in the assay (LOQ) and 1 ng/mg. In some embodiments, the recombinant polypeptide is selected from a growth factor, a cytokine, an antibody, an antibody fragment, and an immunoadhesin. In some embodiments, the recombinant polypeptide is an antibody. In some embodiments, the antibody is a humanized monoclonal antibody. In some embodiments, the antibody is IgG1 or IgG2 or IgG3 or IgG4. In some embodiments, the antibody is IgG1. In some embodiments, the antibody is IgG2. In some embodiments, the antibody is IgG3. In some embodiments, the antibody is IgG4. In some embodiments, the method includes a hydrophobic interaction chromatography (HIC) step. In some embodiments, the HIC step includes the use of PHENYL SEPHAROSE ^TM 6 Fast Flow (High Sub) resin.

In some embodiments of the above purification methods, the purified antibody is anti-IL13. In some embodiments, the antibody is lebrikizumab. In some embodiments, the HIC step includes using a resin column in breakthrough collection mode. In some embodiments, the HIC step includes the use of an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer comprise 50 mM sodium acetate, pH 5.0. In some embodiments, breakthrough is monitored by absorbance at 280 nanometers and breakthrough is collected between 0.5 OD and 1.5 OD. In some embodiments, the breakthrough is collected up to a maximum of 8 column volumes. In some embodiments, the method further includes an affinity chromatography step. In some embodiments, the affinity chromatography is protein A chromatography. In some embodiments, the method further includes an ion exchange chromatography step. In some embodiments, the ion exchange chromatography is an anion exchange chromatography. In some embodiments, the methods include a first step protein A affinity chromatography, a second step anion exchange chromatography, and a third step hydrophobic interaction chromatography (HIC). In some embodiments, the affinity chromatography step includes the use of MABSELECT SURE ^TM resin, the anion exchange chromatography step includes the use of Q SEPHAROSE ^TM Fast Flow resin, and the HIC step includes the use of PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) resin. In some embodiments, the affinity chromatography step includes the use of a MABSELECT SURE ^™ resin column in binding and elution mode, the anion exchange chromatography step includes the use of a Q SEPHAROSE ^™ Fast Flow resin column in the binding and elution mode, and the HIC step includes the use of a PHENYL resin column SEPHAROSE ^TM 6 Fast Flow (High Sub) in breakthrough mode. In some embodiments, the amount of hamster PLBL2 is quantified using immunoassay or mass spectrometric analysis. In some embodiments, the immunoassay is a Chinese Hamster Ovary all protein ELISA or a hamster PLBL2 ELISA. In some embodiments, the mass spectrometry analysis is LC-MS/MS.

In some embodiments of the above purification methods, the purified antibody is an anti-Abeta. In some embodiments, the anti-Abeta antibody is crenezumab. In some embodiments, the anti-Abeta antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.:23, CDR-H2 having the amino acid sequence of SEQ ID NO.:24, and CDR-H3 having the amino acid sequence the sequence of SEQ ID NO.:25, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.:26, CDR-L2 having the amino acid sequence of SEQ ID NO.:27, and CDR-L3 having the amino acid sequence SEQ ID NO.:28. In some embodiments, the anti-Abeta antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.:29. In some embodiments, the anti-Abeta antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO.:30. In some embodiments, the anti-Abeta antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.:29 and a light chain variable region having the amino acid sequence of SEQ ID NO.:30. In some embodiments, the HIC step includes using a resin column in breakthrough collection mode. In some embodiments, the HIC step comprises the use of an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer comprise 150 mM sodium acetate pH 5.0. In some embodiments, the HIC step includes the use of an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer comprise 150 mM sodium acetate pH 4.0. In some embodiments, the HIC step comprises the use of an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer comprise 150 mM sodium acetate, 240 mM sodium sulfate, pH 4.0. In some embodiments, the HIC step comprises the use of an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer comprise 150 mM sodium acetate, 240 mM sodium sulfate, pH 5.0. In some embodiments, the application density is 300 g/l. In some embodiments, the application density is 100 g/l. In some embodiments, the breakthrough is monitored by absorbance at 280 nanometers and the breakthrough is collected starting at 0.5 OD and in a volume of 10 column volumes. In some embodiments, the method further includes an affinity chromatography step. In some embodiments, the affinity chromatography is protein A chromatography. In some embodiments, the methods further include a combined chromatography step. In some embodiments, the methods include a first step protein A affinity chromatography, a second step combined chromatography, and a third step hydrophobic interaction chromatography (HIC). In some embodiments, the affinity chromatography step includes the use of MABSELECT SURE ^TM resin, the combined chromatography step includes the use of CAPTO ^TM Adhere resin, and the HIC step includes the use of PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) resin. In some embodiments, the affinity chromatography step includes the use of a MABSELECT SURE ^TM resin column in binding and elution mode, the combined chromatography step includes the use of a CAPTO ^TM Adhere resin column in the breakthrough mode, and the HIC step includes the use of a PHENYL SEPHAROSE ^TM 6 resin column Fast Flow (High Sub) in breakthrough mode. In some embodiments, the amount of hamster PLBL2 is quantified using immunoassay or mass spectrometric analysis. In some embodiments, the immunoassay is a Chinese Hamster Ovary all protein ELISA or a hamster PLBL2 ELISA. In some embodiments, the mass spectrometry analysis is LC-MS/MS.

In another additional aspect of the above purification methods, the purified antibody is IgG1. In some embodiments, the antibody is anti-IL17 A/F. In some embodiments, the anti-IL17 A/F antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.:15, CDR-H2 having the amino acid sequence of SEQ ID NO.:16, and CDR-H3 having the amino acid sequence of SEQ ID NO.:17 and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.:18, CDR-L2 having the amino acid sequence of SEQ ID NO.:19, and CDR-L3, having the amino acid sequence of SEQ ID NO.:20. In some embodiments, the anti-IL17 A/F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.:21. In some embodiments, the anti-IL17 A/F antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO.:22. In some embodiments, the anti-IL17 A/F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.:21 and a light chain variable region having the amino acid sequence of SEQ ID NO.:22. In some embodiments, the HIC chromatography step includes the use of an equilibration buffer and wash buffer, where the equilibration buffer and wash buffer comprise 50 mM sodium acetate, pH 5.5. In some embodiments, the breakthrough is monitored by absorbance at 280 nanometers and the breakthrough is collected starting at 0.5 OD and in a volume of 10 column volumes. In some embodiments, the method further includes an affinity chromatography step. In some embodiments, the affinity chromatography is Protein A chromatography. In some embodiments, the methods further include a cation exchange chromatography step. In some embodiments, the methods include a first affinity chromatography step and a second cation exchange chromatography step before the hydrophobic interaction chromatography step. In some embodiments, the affinity chromatography step includes the use of MABSELECT SURE ^TM resin, the cation exchange chromatography step includes the use of POROS 50 HS resin, and the HIC step includes the use of PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) resin. In some embodiments, the affinity chromatography step includes using a MABSELECT SURE ^TM resin column in bind and elution mode; the cation exchange chromatography step includes the use of a PORO 50 HS resin column in the binding and elution mode, and the HIC step includes the use of a PHENYL SEPHAROSE ^TM 6 Fast Flow (High Sub) resin column in the breakthrough mode.

In yet another aspect, monoclonal anti-Abeta formulations are provided, antibodies purified from CHO cells by a process including a hydrophobic interaction chromatography (HIC) step. In some embodiments, the purified preparation comprises an anti-Abeta antibody and a residual amount of hamster PLBL2. In some embodiments, the amount of hamster PLBL2 is less than 20 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 15 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 10 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 8 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 5 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 3 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 2 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 1 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 0.5 ng/mg. In some embodiments, the amount of hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg. mg, or between 0.5 ng/mg and 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3 ng/mg, or between 0.5 ng /mg and 2 ng/mg, or between 0.5 ng/mg and 1 ng/mg, or between the limit of quantification in the assay (LOQ) and 1 ng/mg. In some embodiments, the HIC step includes the use of PHENYL SEPHAROSE ^TM 6 Fast Flow (High Sub) resin. In some embodiments, the HIC step includes using a resin column in breakthrough collection mode. In some embodiments, the HIC step comprises the use of an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer comprise 150 mM sodium acetate, pH 5.0. In some embodiments, the HIC step comprises the use of an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer comprise 150 mM sodium acetate, pH 4.0. In some embodiments, the HIC step comprises the use of an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer comprise 150 mM sodium acetate, 240 mM sodium sulfate, pH 4.0. In some embodiments, the HIC step comprises the use of an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer comprise 150 mM sodium acetate, 240 mM sodium sulfate, pH 5.0. In some embodiments, the application density is 300 g/l. In some embodiments, the application density is 100 g/l. In some embodiments, the breakthrough is monitored by absorbance at 280 nanometers and the breakthrough is collected starting at 0.5 OD and in a volume of 10 column volumes. In some embodiments, the process further includes an affinity chromatography step. In some embodiments, the affinity chromatography is protein A chromatography. In some embodiments, the process further includes a combined chromatography step. In some embodiments, the anti-Abeta antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.: 23, CDR-H2 having the amino acid sequence of SEQ ID NO.: 24, and CDR-H3 having the amino acid sequence sequence of SEQ ID NO.: 25, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.: 26, CDR-L2 having the amino acid sequence of SEQ ID NO.: 27, and CDR-L3 having the amino acid sequence SEQ ID NO.: 28. In some embodiments, the anti-Abeta antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 29. In some embodiments, the anti-Abeta antibody comprises a light chain variable region having the amino acid sequence SEQ ID NO.: 30. In some embodiments, the anti-Abeta antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 29 and a light chain variable region having the amino acid sequence of SEQ ID NO.: 30. In some embodiments, the amount of hamster PLBL2 is quantified using immunoassay or mass spectrometric analysis. In some embodiments, the immunoassay is a Chinese Hamster Ovary all protein ELISA or a hamster PLBL2 ELISA. In some embodiments, the mass spectrometry analysis is LC-MS/MS.

In one aspect, anti-IL17 A/F monoclonal antibody preparations are provided that are isolated and purified from CHO cells by a process including a hydrophobic interaction chromatography (HIC) step. In some embodiments, the purified preparation comprises an anti-IL17 A/F antibody and a residual amount of hamster PLBL2. In some embodiments, the amount of hamster PLBL2 is less than 20 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 15 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 10 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 8 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 5 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 3 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 2 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 1 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 0.5 ng/mg. In some embodiments, the amount of hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg. mg, or between 0.5 ng/mg and 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3 ng/mg, or between 0.5 ng /mg and 2 ng/mg, or between 0.5 ng/mg and 1 ng/mg, or between the limit of quantification in the assay (LOQ) and 1 ng/mg. In some embodiments, the HIC step includes the use of PHENYL SEPHAROSE ^TM 6 Fast Flow (High Sub) resin. In some embodiments, the HIC step includes using a resin column in breakthrough collection mode. In some embodiments, the HIC step includes the use of an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer comprise 50 mM sodium acetate, pH 5.5. In some embodiments, the breakthrough is monitored by absorbance at 280 nanometers and the breakthrough is collected starting at 0.5 OD and in a volume of 10 column volumes. In some embodiments, the process further includes an affinity chromatography step. In some embodiments, the affinity chromatography is protein A chromatography. In some embodiments, the process further includes a cation exchange chromatography step. In some embodiments, the anti-IL17 A/F antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.: 15, CDR-H2 having the amino acid sequence of SEQ ID NO.: 16, and CDR-H3 , having the amino acid sequence of SEQ ID NO.: 17, and three light chain CDRs, CDR-L1, having the amino acid sequence of SEQ ID NO.: 18, CDR-L2, having the amino acid sequence of SEQ ID NO.: 19, and CDR-L3, having the amino acid sequence of SEQ ID NO.: 20. In some embodiments, the anti-IL17 A/F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 21. In some embodiments, the anti-IL17 A/F antibody includes a light chain variable region having the amino acid sequence of SEQ ID NO.: 22. In some embodiments, an anti-IL17 A/F antibody includes a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 21 and a light chain variable region, having the amino acid sequence of SEQ ID NO.: 32. In some embodiments, the amount of hamster PLBL2 is quantified using immunoassay or mass spectrometric analysis. In some embodiments, the immunoassay is a Chinese Hamster Ovary all protein ELISA or a hamster PLBL2 ELISA. In some embodiments, the mass spectrometry analysis is LC-MS/MS.

In yet another aspect, compositions are provided comprising an anti-Abeta monoclonal antibody purified from CHO cells, comprising an anti-Abeta antibody and a residual amount of hamster PLBL2. In some embodiments, the amount of hamster PLBL2 is less than 20 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 15 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 10 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 8 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 5 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 3 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 2 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 1 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 0.5 ng/mg. In some embodiments, the amount of hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg. mg, or between 0.5 ng/mg and 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3 ng/mg, or between 0.5 ng /mg and 2 ng/mg, or between 0.5 ng/mg and 1 ng/mg, or between the limit of quantification in the assay (LOQ) and 1 ng/mg. In some embodiments, the anti-Abeta antibody is crenezumab. In some embodiments, the anti-Abeta antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.:23, CDR-H2 having the amino acid sequence of SEQ ID NO.:24, and CDR-H3 having the amino acid sequence the sequence of SEQ ID NO.:25, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.:26, CDR-L2 having the amino acid sequence of SEQ ID NO.:27, and CDR-L3 having the amino acid sequence SEQ ID NO.:28. In some embodiments, the anti-Abeta antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.:29. In some embodiments, the anti-Abeta antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO.:30. In some embodiments, the anti-Abeta antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.:29 and a light chain variable region having the amino acid sequence of SEQ ID NO.:30.

In yet another aspect, compositions are provided comprising an anti-IL17 A/F monoclonal antibody purified from CHO cells, comprising an anti-IL17 A/F antibody and a residual amount of hamster PLBL2. In some embodiments, the composition comprises an anti-IL17 A/F antibody and a residual amount of hamster PLBL2, wherein the amount of hamster PLBL2 is less than 20 ng/mg, or less than 15 ng/mg, or less than 10 ng/mg, or less than 8 ng/mg. mg, or less than 5 ng/mg, or less than 3 ng/mg, or less than 2 ng/mg, or less than 1 ng/mg, or less than 0.5 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 20 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 15 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 10 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 8 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 5 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 3 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 2 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 1 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 0.5 ng/mg. In some embodiments, the amount of hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg. mg, or between 0.5 ng/mg and 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3 ng/mg, or between 0.5 ng /mg and 2 ng/mg, or between 0.5 ng/mg and 1 ng/mg, or between the limit of quantification in the assay (LOQ) and 1 ng/mg. In some embodiments, the anti-IL17 A/F antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.:15, CDR-H2 having the amino acid sequence of SEQ ID NO.:16, and CDR-H3 having the amino acid sequence of SEQ ID NO.:17 and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.:18, CDR-L2 having the amino acid sequence of SEQ ID NO.:19, and CDR-L3, having the amino acid sequence of SEQ ID NO.:20. In some embodiments, the anti-IL17 A/F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.:21. In some embodiments, the anti-IL17 A/F antibody comprises a light chain variable region having the amino acid sequence of SEQ ID NO.:22. In some embodiments, the anti-IL17 A/F antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.:21 and a light chain variable region having the amino acid sequence of SEQ ID NO.:22.

In one aspect, methods of treating an IL-13 mediated disorder are provided, comprising administering a treatment composition comprising an anti-IL13 monoclonal antibody purified from CHO cells and a residual amount of hamster PLBL2. In some embodiments, the amount of hamster PLBL2 is less than 20 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 15 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 10 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 8 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 5 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 3 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 2 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 1 ng/mg. In some embodiments, the amount of hamster PLBL2 is less than 0.5 ng/mg. In some embodiments, the amount of hamster PLBL2 is between 0.5 ng/mg and 20 ng/mg, or between 0.5 ng/mg and 15 ng/mg, or between 0.5 ng/mg and 10 ng/mg. mg, or between 0.5 ng/mg and 8 ng/mg, or between 0.5 ng/mg and 5 ng/mg, or between 0.5 ng/mg and 3 ng/mg, or between 0.5 ng /mg and 2 ng/mg, or between 0.5 ng/mg and 1 ng/mg, or between the limit of quantification in the assay (LOQ) and 1 ng/mg. In some embodiments, the anti-IL13 antibody comprises three heavy chain CDRs, CDR-H1 having the amino acid sequence of SEQ ID NO.: 1, CDR-H2 having the amino acid sequence of SEQ ID NO.: 2, and CDR-H3 having the amino acid sequence sequence of SEQ ID NO.: 3, and three light chain CDRs, CDR-L1 having the amino acid sequence of SEQ ID NO.: 4, CDR-L2 having the amino acid sequence of SEQ ID NO.: 5, and CDR-L3 having the amino acid sequence SEQ ID NO.: 6. In some embodiments, the anti-IL13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 7. In some embodiments, the anti-IL13 antibody comprises a light chain variable region having the amino acid sequence SEQ ID NO.: 9. In some embodiments, the anti-IL13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO.: 10. In some embodiments, the anti-IL13 antibody comprises a light chain having the amino acid sequence of SEQ ID NO. : 14. In some embodiments, the anti-IL13 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO.: 7 and a light chain variable region having the amino acid sequence of SEQ ID NO.: 9. In some embodiments, the anti -IL13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO.: 10 and a light chain having the amino acid sequence of SEQ ID NO.: 14. In some embodiments, the treatment composition is administered subcutaneously once every four weeks. In some embodiments, the treatment composition is administered subcutaneously once every eight weeks. In some embodiments, the treatment composition is administered subcutaneously once every 12 weeks. In some embodiments, the patient is treated once every four weeks for at least one month. In some embodiments, the patient is treated once every four weeks for at least three months. In some embodiments, the patient is treated once every four weeks for at least six months. In some embodiments, the patient is treated once every four weeks for at least nine months. In some embodiments, the patient is treated once every four weeks for at least 12 months. In some embodiments, the patient is treated once every four weeks for at least 18 months. In some embodiments, the patient is treated once every four weeks for at least two years. In some embodiments, the patient is treated once every four weeks for more than two years. In some embodiments, the IL-13 mediated disorder is asthma. In some embodiments, the IL-13 mediated disorder is idiopathic pulmonary fibrosis. In some embodiments, the IL-13 mediated disorder is atopic dermatitis. In some embodiments, the IL-13 mediated disorder is selected from allergic asthma, non-allergic (endogenous) asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, eczema, urticaria, food allergies, chronic obstructive pulmonary disease, ulcerative colitis, RSV infection, uveitis, scleroderma and osteoporosis.

In another aspect, administering a treatment composition to a patient according to any method described above is less immunogenic for hamster PLBL2 compared to administering a reference composition, wherein the reference composition comprises an anti-IL13 monoclonal antibody purified from Chinese hamster ovary host cells, and residual amount of hamster PLBL2, the amount of which is greater than 30 ng/mg. In some embodiments, the amount of hamster PLBL2 in the reference composition is greater than 50 ng/mg. In some embodiments, the amount of hamster PLBL2 in the reference composition is greater than 100 ng/mg. In some embodiments, the amount of hamster PLBL2 in the reference composition is greater than 200 ng/mg. In some embodiments, the amount of hamster PLBL2 in the reference composition is greater than 300 ng/mg. In some embodiments, the amount of hamster PLBL2 in the reference composition is between 30 ng/mg and 300 ng/mg, or between 30 ng/mg and 200 ng/mg, or between 30 ng/mg and 100 ng/mg, or between 30 ng/mg and 50 ng/mg.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows CHOP levels in anti-IL13 MAb pools made using protein A treated with caprylic acid as described in Example 2. (A) Precipitation of a pool made using protein A with caprylic acid at pH 4.5; (B) Precipitation of the pool obtained using A with caprylic acid at pH 5.0. CHOP levels in ng/mg are marked along the vertical axis; the percentage of caprylic acid is marked along the horizontal axis, each bar represents the value obtained from the 2-fold serial dilution.

Figure 2 shows total CHOP levels in HCCF-supplemented anti-IL13 MAb followed by protein A chromatography followed by POROS® 50HS cation exchange chromatography as described in Example 2. Adjusted CHOP levels in ng/mL are plotted on the vertical axis; additives (control, 0.6M guanidine or 0.6M arginine) are indicated on the horizontal axis, each bar represents the value obtained from 2-fold serial dilution as indicated.

Figure 3 shows total CHOP levels in anti-IL13 MAb UFDF pools purified on different HIC resins at different salt concentrations and pH values as described in Example 2. (A) OCTYL-SEPHAROSE® Fast Flow resin; (B) PHENYL SEPHAROSE ^TM 6 Fast Flow (low sub) resin; (C) BUTYL-SEPHAROSE® 4 Fast Flow resin; (D) PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) resin; the highest dilution of CHOP (in ppm) is indicated on the vertical axis, and sodium sulfate concentrations are noted on the horizontal axis; pH (5.5, 6.0, 7.0 or 8.0) is indicated in the captions to the Figure.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which this invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994) and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992) are a general guide for a person skilled in the art for many of the terms used in this application.

SOME DEFINITIONS

For the purpose of interpreting this specification, the following definitions will apply and, whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition below is inconsistent with that of any of the documents incorporated herein by reference, the definition below shall be taken as the primary definition.

As used in this specification and the appended claims, the singular forms also include the plural, unless the context clearly indicates otherwise. Thus, for example, reference to "protein" or "antibody" includes a plurality of proteins or antibodies, respectively; reference to "cell" includes a mixture of cells and the like.

The term "detection" is used herein in a broad sense and includes both qualitative and quantitative measurement of the target molecule. Detection involves only determining whether the target molecule is present in the sample, as well as determining whether the target molecule is present in the sample in a detectable amount.

"Sample" refers to a small portion of a larger amount of material. Typically, testing in accordance with the methods described herein is carried out using a sample. The sample is typically obtained from a preparation of a recombinant polypeptide obtained, for example, from cultured host cells. The sample can be obtained, for example, from the collected culture liquid, from the in-house pool taken at a certain stage of the purification process, or from the final purified product, but not limited to the above.

The term "product", as described herein, is a substance that must be purified by various chromatographic methods; for example, a polypeptide.

The terms "polypeptide" or "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, may include modified amino acids, and may also include non-amino acids. The terms further include an amino acid polymer that has been naturally or tampered with; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation to a labeled moiety. This definition also includes, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids and the like), as well as other modifications known in the art. As used herein, the terms "polypeptide" and "protein" specifically include antibodies.

A "purified" polypeptide (for example, an antibody or immunoadhesin) means that the purity of the polypeptide has been increased such that the polypeptide exists in a purer form than it exists in the natural environment and/or compared to the originally synthesized and/or or a laboratory-amplified form. Purity is a relative term and does not necessarily mean absolute purity.

As used herein, the term "epitope-tagged" refers to a chimeric polypeptide comprising a polypeptide fused to a "tag polypeptide". The number of residues in the tag polypeptide is sufficient to provide an epitope against which an antibody can be generated, but at the same time the tag polypeptide is short enough not to affect the activity of the polypeptide to which it is fused. The tag polypeptide is preferably also sufficiently unique such that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides typically have at least six amino acid residues, typically about 8 to 50 amino acid residues (in some cases, about 10 to 20 amino acid residues).

The terms "active" or "activity" for the purposes of this document refers to the form(s) of a polypeptide that retains the intended biological and/or immunological activity, where "biological" activity refers to the biological function (inhibitory or stimulatory) due to the polypeptide, but different on its ability to induce the production of an antibody against an antigenic epitope on a polypeptide, and "immunological" activity refers to the ability of a polypeptide to induce the production of an antibody against an antigenic epitope on a polypeptide.

The term "antagonist" is used herein in a broad sense and includes any molecules that partially or completely block, inhibit or neutralize the biological activity of a natural polypeptide, such as a cytokine. Likewise, the term "agonist" is used herein in a broad sense and includes any molecule that mimics the biological activity of a naturally occurring polypeptide. Suitable agonist or antagonist molecules particularly include antibodies or antibody fragments, amino acid sequence fragments or variants of naturally occurring polypeptides, and the like. Methods for identifying agonists or antagonists of a polypeptide may include contacting the polypeptide with a potential agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide.

A polypeptide "that binds to" a target antigen, e.g., a tumor-associated target polypeptide antigen, is a polypeptide that binds to the antigen with sufficient affinity such that the polypeptide is used as an assay reagent, diagnostic and/or therapeutic agent for targeted interaction. with a sample containing an antigen, cells or tissues expressing the antigen, and does not have significant cross-reactivity with other polypeptides.

In relation to the binding of a polypeptide to a target molecule, the terms "specific binding" to a particular polypeptide or epitope on a particular target polypeptide, or "specifically binds to" a particular polypeptide or epitope on a particular target polypeptide, or "specific to" a particular polypeptide or epitope on a particular target polypeptide means a binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by comparing the binding of a molecule and the binding of a control molecule, which is usually a molecule having a similar structure but no binding activity. For example, specific binding can be determined by competition for binding to a control molecule similar to the target molecule, eg using an excess of unlabeled target molecule. In this case, specific binding is present if binding of the labeled target molecule to the probe is competitively inhibited by an excess of unlabeled target molecule.

The term "antibody" is used herein in a broad sense and includes various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, provided they have the desired biological activity. The term "immunoglobulin" (Ig) and the term "antibody" are used interchangeably herein.

Antibodies are naturally occurring immunoglobulin molecules having various structures and chain folds characteristic of immunoglobulins. For example, IgG antibodies have two "heavy" chains and two "light" chains that are linked by disulfide bonds to form a functional antibody. The heavy and light chains each individually comprise a "constant" (C) and a "variable" (V) region. The V regions determine the antigen-binding specificity of the antibody, while the C regions provide the structural framework of the molecule and are involved in interactions with immune effectors unrelated to antigen specificity. The antigen-binding specificity of an antibody or antigen-binding fragment of an antibody is the ability of an antibody to specifically bind to a particular antigen.

The antigen-binding specificity of an antibody is determined by the structural characteristics of the V region. The variability is not evenly distributed among the 110 amino acids of the variable domains. Conversely, V regions are composed of relatively invariant regions called framework regions (FRs) of 15-30 amino acids separated by shorter highly variable regions called "hypervariable regions" each 9-12 amino acids long. Each native heavy and light chain variable domain comprises four FRs, generally assuming a β-sheet configuration, connected by three hypervariable regions that form loops connecting β-sheet structures and, in some cases, forming part of the β-sheet structure. The hypervariable regions in each strand are held in close proximity to each other by FRs and, together with hypervariable regions from the other strand, contribute to the formation of the antibody antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but are responsible for various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC).

Each V region typically includes three complementarity determining regions ("CDRs", each containing a "hypervariable loop") and four framework regions. Antigen binding site, the minimum structural unit required to bind with significant affinity to a particular target antigen, therefore, will typically include three CDRs and at least three, preferably four, framework regions located between them to hold and present CDR in the appropriate conformation. Classical four-chain antibodies have antigen-binding sites that are defined jointly by the V _H and V _L domains. Some antibodies, such as camel and shark antibodies, lack light chains and have binding sites formed only by heavy chains. Genetically engineered single chain immunoglobulins can be prepared in which binding sites are formed only by heavy chains or only by light chains, in the absence of cooperation between V _H and V _L .

As used herein, the term "hypervariable region" refers to certain amino acid residues in antibodies that are responsible for antigen binding. The hypervariable region may include amino acid residues from the "complementarity determining region" or "CDR" as discussed above (e.g., near residues about 24-34 (L1), 50-56 (L2), and 89-97 (L3) in V _L and near residues approximately 31-35B (H1), 50-65 (H2), and 95-102 (H3) in V _H (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health , Bethesda, Md. (1991)), and/or residues from the "hypervariable loop" (for example, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in V _L and 26-32 (H1), 52A-55 (H2) and 96-101 (H3) in V _H (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

"Frame" or "FR" residues are variable domain residues other than hypervariable region residues as defined herein.

"Antibody fragments" include a portion of an intact antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments; diabody; tandem diabodies (taDb), linear antibodies (eg, US Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); single arm antibodies, single variable domain antibodies, minibodies, single chain antibody molecules; multispecific antibodies formed from antibody fragments (for example, including but not limited to Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, di-scFv, bi-scFv or tandem (di,tri) -scFv); and bispecific T cell activators (BiTE).

Cleavage of antibodies with papain produces two identical antigen-binding fragments, called "Fab" fragments, each with one antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to easily crystallize. Pepsin treatment produces an F(ab') ₂ fragment that has two antigen-binding sites and is still able to cross-link the antigen.

"Fv" is the minimum antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in a tight, non-covalent relationship with each other. It is in this configuration that the three hypervariable regions of each variable domain interact to form an antigen-binding site on the surface of the V _H -V _L dimer. Collectively, the six hypervariable regions determine the antigen-binding specificity of an antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific hypervariable regions) has the ability to recognize and bind an antigen, albeit at a lower affinity than the full binding site.

The Fab fragment further comprises a light chain constant domain and a heavy chain first constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine residues from the antibody hinge region. Fab'-SH as used herein is the designation for a Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group. F(ab') ₂ antibody fragments were originally generated as pairs of Fab' fragments with hinged cysteines in between. Other chemical methods for binding antibody fragments are also known.

"Light chains" of antibodies (immunoglobulins) from any vertebrate species, based on the amino acid sequences of their constant domains, can be assigned to one of two distinct types, called kappa (κ) and lambda (λ).

Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

"Single chain Fv" or "scFv" antibody fragments include the V _H and V _L domains of an antibody, where these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the V _H and V _L domains that allows the scFv to form the desired structure for antigen binding. An overview of scFv can be found in Plückthun, The Pharmacology of Monoclonal antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen binding sites, where the fragments include a heavy chain variable domain (V _H ) connected to a light chain variable domain (V _L ) in the same polypeptide chain (V _H - V _L ). When using a linker that is too short to allow two domains of the same strand to pair with each other, the domains are forced to pair with complementary domains of the other strand and form two antigen-binding sites. Diabodies are described in more detail, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. sci. USA 90:6444-6448 (1993).

The term "multispecific antibody" is used herein in a broad sense and specifically encompasses an antibody that has polyepitope specificity. Such multispecific antibodies include, but are not limited to, an antibody comprising a heavy chain variable domain (V _H ) and a light chain variable domain (V _L ), where the V _H V _L unit has polyepitope specificity, antibodies having two or more V _L domains and V _H , where each V _H V _L unit binds to its epitope, antibodies having two or more single variable domains, where each single variable domain binds to its epitope, full length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, tribodies, tri-functional antibodies, covalently or non-covalently linked antibody fragments. "Polyepitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. The term "monospecific" refers to the ability to bind to only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 μM to 0.001 pM, 3 μM to 0.001 pM, 1 μM to 0.001 pM, 0.5 μM to 0.001 pM or 0.1 µM to 0.001 pM.

The expression "single domain antibodies" (sdAb) or "antibodies with a single variable domain (SVD)" usually refers to antibodies in which a single variable domain (VH or VL) can provide antigen binding. In other words, in order to recognize the target antigen, a single variable domain does not need to interact with another variable domain. Examples of single-domain antibodies include antibodies derived from camelids (llamas and camels) and cartilaginous fish (eg, nurse sharks) and antibodies that are recombinantly derived from human and murine antibodies (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56 Trend Biochem Sci (2001) 26:230-235 Trends Biotechnol (2003):21:484-490 WO 2005/035572 WO 03/035694 Febs Lett (1994) 339 :285-290; WO00/29004; WO 02/051870).

As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical and/or bind to the same epitope, except for possible variants that may arise during the production of a monoclonal antibody, but such variants are usually present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of not being contaminated by other immunoglobulins. The modifier "monoclonal" indicates the nature of the antibody as being derived from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the methods provided herein can be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be made by recombinant DNA methods (see e.g. , US patent number 4,816,567). "Monoclonal antibodies" can also be isolated from antibody phage libraries using the methods described, for example, in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

As used herein, monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remaining part of the chain(s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they have the desired biological activity (US patent number 4,816,567; Morrison et al. , Proc Natl Acad Sci USA 81:6851-6855 (1984)). Targeted chimeric antibodies herein include "primatized" antibodies comprising antigen-binding sequences of variable domains from antibodies of non-human primates (e.g., marmosets such as baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences (U.S. Pat. number 5,693,780).

"Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region of the recipient are replaced with residues from the hypervariable region of a non-human antibody (donor antibody), such as a mouse, rat, rabbit, or primate antibody, non-human having the desired specificity, affinity and capacity. In some cases, framework region (FR) residues of a human immunoglobulin are replaced with corresponding non-human residues. Moreover, humanized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the activity of the antibody. In general, a humanized antibody will contain substantially all of at least one and typically two variable domains wherein all or substantially all of the hypervariable loops correspond to those in non-human immunoglobulins and all or substantially all of the FRs are is a FR from a human immunoglobulin sequence, except for the substitution(s) in the FR mentioned above. The humanized antibody will optionally further include at least a portion of an immunoglobulin constant region, typically a human immunoglobulin. For more information, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

For the purposes of this document, a "tact antibody" is an antibody comprising heavy and light chain variable domains and an Fc region. The constant domains may have a sequence of native constant domains (eg, the sequence of human native constant domains) or be a variant of their amino acid sequence. Preferably, the intact antibody has one or more effector functions.

"Native antibodies" are usually heterotetrameric glycoproteins having a size of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by a single covalent disulfide bond, while the number of disulfide bonds between heavy chains may vary for different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain contains at one end a variable domain (V _H ) followed by several constant domains. Each light chain has a variable domain (V _L ) at one end and a constant domain at the other end; the light chain constant domain is aligned with the first heavy chain constant domain, and the light chain variable domain is aligned with the heavy chain variable domain. Certain amino acid residues are thought to form a region of contact between the light chain and heavy chain variable domains.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence, after aligning sequences and introducing breaks, if necessary, to obtain the maximum percent sequence identity and ignoring any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR ). Those of skill in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to obtain maximum alignment between compared full-length sequences. For the purposes of this document, however, % amino acid sequence identity values are obtained using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc. and its source code has been filed with the user documentation at the United States Copyright Office, Washington, D.C. 20559, where it has been registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from source code. The ALIGN-2 program must be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All parameters for sequence comparison are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is used to compare amino acid sequences, the % amino acid sequence identity for a given amino acid sequence A and a given amino acid sequence B (can alternatively be paraphrased as: a given amino acid sequence A shares some % or some % amino acid sequence identity with a given amino acid sequence). sequence B) is calculated as follows:

100 times X/Y,

where X is the number of amino acid residues that are identical matches, as determined by the ALIGN-2 sequence alignment program when aligning A and B, and where Y is the total number of amino acid residues in B. It should be understood that where the length the amino acid sequence of A is not equal to the length of the amino acid sequence of B, the % amino acid sequence identity of A with B will not be equal to the % amino acid sequence identity of B with A. Unless otherwise noted, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The terms "anti-IL-13 antibody" and "antibody that binds to IL-13" refer to an antibody that is capable of binding to IL-13 with sufficient affinity to enable the antibody to be used as a diagnostic and/or therapeutic agent for targeted interaction with IL-13. In some embodiments, the degree of binding of an anti-IL-13 antibody to an unrelated non-IL-13 protein is less than about 10% of the binding of the antibody to IL-13, as measured by, for example, radioimmunoassay (RIA). In some embodiments, the antibody that binds to IL-13 has a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (for example, 10 ^-8 M or less, for example, from 10 ^-8 M to 10 ^-13 M, for example, from 10 ^-9 M to 10 ^-13 M). In some embodiments, the anti-IL-13 antibody binds to an IL-13 epitope that is species-conserved.

"IL-13 mediated disorder" means a disorder associated with excessive levels of IL-13 or its excessive activity, in which atypical symptoms are manifested due to high levels or activity of IL-13 locally and/or in the body as a whole. Examples of IL-13 mediated disorders include cancer (eg, non-Hodgkin's lymphoma, glioblastoma), atopic dermatitis, allergic rhinitis, asthma, fibrosis, inflammatory bowel disease, Crohn's disease, inflammatory disorders of the lungs (including pulmonary fibrosis, eg, IPF), COPD, and fibrosis of the liver.

The term "respiratory disorder" includes asthma (eg, allergic and non-allergic asthma (eg, due to infection, eg, caused by respiratory syncytial virus (RSV), eg, in young children)); bronchitis (eg, chronic bronchitis); chronic obstructive pulmonary disease (COPD) (e.g., emphysema (e.g., smoking-induced emphysema); conditions including, but not limited to, airway inflammation, eosinophilia, fibrosis, and excessive mucus production, such as, but not limited to, cystic fibrosis, pulmonary fibrosis, and allergic rhinitis. Examples diseases that can be characterized by inflammation of the airways, excessive secretions in the airways and airway obstruction include asthma, chronic bronchitis, bronchial ecstasy and cystic fibrosis.

The term "therapeutic agent" refers to any agent that is used to treat a disease. The therapeutic agent may be, for example, a polypeptide(s) (e.g., an antibody, an immunoadhesin, or a peptibody), an aptamer, or a small molecule that can bind to a protein or nucleic acid molecule encoding a target (i.e., siRNA) and similar.

A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic fragment or a radioactive label.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformers" and "transformed cells", which include the primary transformed cell and its descendants, regardless of the number of passages. The nucleic acids contained in the descendants of the cell may not be completely identical to the nucleic acids contained in the parent cell, as they may contain mutations. Mutant progeny that exhibit the same function or biological activity that was selected or sought by screening from the originally transformed cells are also included herein.

As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid to which it is linked. The term includes a vector as a self-replicating nucleic acid structure, as well as a vector inserted into the genome of the host cell into which it has been introduced. Some vectors are capable of inducing expression of the nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

An "isolated" antibody is an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is greater than 95% or 99% pure, where purity is determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectrofocusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The term "sequential" as used herein in relation to chromatography means that a second chromatography follows the first chromatography. Additional steps may be included between the first chromatography and the second chromatography.

The term "continuous" as used herein in relation to chromatography means that the first chromatographic material and the second chromatographic material are connected either directly or through some other mechanism that provides a continuous flow between the two chromatographic materials.

"Impurities" and "contaminants" refer to materials that differ from the desired polypeptide product. Impurities and contaminants include, without limitation, host cell materials such as CHOP, including CHOP of a single species; leached protein A; nucleic acid; variant, fragment, aggregate or derivative of the target polypeptide; another polypeptide; endotoxin; viral contaminant; component of the cultural environment and the like. In some examples, the contaminant may be a host cell protein (HCP), e.g., a bacterial cell such as an E. coli cell, an insect cell, a prokaryotic cell, a eukaryotic cell, a yeast cell, a mammalian cell, a bird cell, a fungus cell, but not limited to them.

The terms "Chinese hamster ovary cell protein" and "CHOP" are used interchangeably and refer to a mixture of host cell proteins ("HCP") derived from a culture of Chinese hamster ovary ("CHO") cells. The HCP or CHOP is usually present as impurities in the culture medium or lysate (eg, harvested culture fluid ("HCCF")) comprising the target protein, such as an antibody or immunoadhesin expressed in the CHO cell. The amount of CHOP present in a mixture containing the target protein is an indication of the degree of purity of the target protein. HCP or CHOP include, but are not limited to, a target protein expressed by a host cell such as a CHO host cell. Typically, the amount of CHOP in a protein mixture is expressed in parts per million compared to the amount of target protein in the mixture. It is understood that if the host cell is another type of mammalian cell, an E. coli cell, a yeast cell, an insect cell, or a plant cell, HCP refers to proteins other than the target protein found in the host cell lysate.

The terms "parts per million" or "ppm" are used interchangeably herein and refer to the degree of purity of the target protein, purified using the method according to this document. The ppm units refer to the amount of HCP or CHOP in nanograms/milliliter to the amount of target protein in milligrams/milliliter (i.e., CHOP ppm = (CHOP, ng/mL)/(target protein, mg/mL), where the proteins are in solution). If proteins are dry (eg, lyophilized), ppm refers to (CHOP, ng)/(target protein, mg). The amount of impurities can also be expressed in "ng/mg", which is used interchangeably with ppm.

"Purification" of a polypeptide from a composition comprising a polypeptide and one or more impurities means increasing the purity of the polypeptide in the composition by removing (in whole or in part) at least one impurity from the composition.

A "purification step" may be part of an overall purification process that results in a "homogeneous" composition, which is used herein to refer to a composition containing less than 100 ppm HCP (100 ng/mg) in a composition comprising a target protein, or less 90 ppm (90 ng/mg) or less than 80 ppm (80 ng/mg) or less than 70 ppm (70 ng/mg) or less than 60 ppm (60 ng/mg) or less than 50 ppm 50 ng/mg ), or less than 40 ppm (40 ng/mg), or less than 30 ppm (30 ng/mg), or less than 20 ppm (20 ng/mg), or less than 10 ppm (10 ng/mg), or less than 5ppm ( 5 ng/mg), or less than 3ppm (3 ng/mg) or less than 1 ppm (1 ng/mg). In some embodiments, the HCP is a single species HCP. In one embodiment, the single species HCP is hamster PLBL2.

As used herein, a "composition" to be purified includes the target polypeptide and one or more impurities or contaminants. The composition may be "partially purified" (i.e., the composition has been subjected to one or more purification steps) or may be obtained directly from the host cell or organism producing the polypeptide (e.g., the composition may include harvested culture fluid).

The terms "protein A" and "ProA" are used interchangeably herein and include protein A obtained from its natural source, protein A obtained synthetically (for example, using peptide synthesis or recombinant methods), and its variants that retain the ability to bind with proteins having a CH2/CH3 region such as an Fc region. Protein A is commercially available from various sources. Protein A is usually immobilized on a solid phase support material. The term "ProA" also refers to an affinity chromatography resin or column containing a solid phase chromatography matrix to which Protein A is covalently attached.

The term "chromatography" refers to a process in which a target solute is separated from other solutes in a mixture as a result of differences in the rates at which the individual solutes of the mixture migrate through the stationary phase under the influence of the mobile phase, or in binding and elution processes.

The terms "affinity chromatography" and "protein affinity chromatography" are used interchangeably herein and refer to a protein separation technique in which a target protein or target antibody binds reversibly and specifically to a biospecific ligand. Typically, the biospecific ligand is covalently attached to the chromatographic solid phase material and is available to the target protein in solution as the solution contacts the chromatographic solid phase material. The target protein (e.g., antibody, enzyme, or receptor protein) remains specifically bound with a certain affinity to a biospecific ligand (e.g., antigen, substrate, cofactor, or hormone) during chromatography, while other solutes and/or proteins in solution do not show a noticeable or specific binding to a ligand. Binding of the target protein to the immobilized ligand allows protein contaminants or protein contaminants to pass through the chromatographic column while the target protein remains bound to the immobilized ligand on the solid phase material. The specifically bound target protein is then removed in active form from the immobilized ligand using low pH, high pH, high salt concentration, competitive ligand, and the like, and it passes through the chromatography column with elution buffer, without contaminating proteins or protein impurities that have already passed. through the column before. Any component, such as an antibody, can be used as a ligand to purify the corresponding specific binding protein.

The terms "non-affinity chromatography" and "non-affinity purification" refer to a purification process that does not use affinity chromatography. Non-affinity chromatography includes chromatographic techniques based on non-specific interactions between a target molecule (such as a protein, eg an antibody) and a solid phase matrix.

The term "specific binding" as used herein in the context of chromatography to describe interactions between a target molecule and a ligand bound to a solid phase matrix refers to the generally reversible binding of a target protein to a ligand due to the combined effects of the steric complementarity of the protein and the structures of the ligands at the binding site, combined with electrostatic forces, hydrogen bonds, hydrophobic forces and/or van der Waals forces at the binding site. The higher the spatial complementarity and the stronger the other forces at the binding site, the higher will be the binding specificity of the protein to its respective ligand. Non-limiting examples of specific binding include antibody-antigen binding, enzyme-substrate binding, enzyme-cofactor binding, metal ion chelation, DNA-binding protein-DNA binding, regulatory protein-protein interactions, and the like. Typically, specific binding affinity in affinity chromatography is about 10 ^-4 to 10 ^-8 M free in solution.

The term "non-specific binding" as used herein in the context of chromatography to describe interactions between a target molecule and a ligand or other compound bound to a solid matrix refers to the binding of a target protein to a ligand or compound on a solid phase matrix by electrostatic forces, hydrogen bonds, hydrophobic forces and/or van der Waals forces at the site of interaction, but not at the expense of structural complementarity, which amplifies the effects of forces unrelated to the structure. Examples of non-specific interactions include, but are not limited to, electrostatic, hydrophobic, and van der Waals forces, as well as hydrogen bonds.

"Salt" is a compound resulting from the reaction between an acid and a base. Examples of salts include but are not limited to acetate (eg sodium acetate), citrate (eg sodium citrate), chloride (eg sodium chloride), sulfate (eg sodium sulfate), or potassium salt.

As used herein, the term "solvent" refers to a liquid substance that can dissolve or disperse one or more other substances to form a solution. Solvents include aqueous and organic solvents, where some organic solvents include a non-polar solvent, ethanol, methanol, isopropanol, acetonitrile, hexylene glycol, propylene glycol and 2,2-thiodiglycol.

The term "detergent" refers to ionic and non-ionic surfactants such as polysorbates (eg polysorbates 20 or 80); poloxamers (eg poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octylglycoside; lauryl-, myristyl-, linoleyl- or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl- or cetyl-betaine; lauroamidopropyl, cocamidopropyl, linoleamidopropyl, myristamidopropyl, palmidopropyl or isosteramidopropyl betaine (eg lauroamidopropyl); myristamidopropyl-, palmidopropyl- or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and surfactants of the MONAQUAT ^TM series (Mona Industries, Inc., Paterson, NJ), a polysorbate such as polysorbate 20 (TWEEN 20®) or polysorbate 80 (TWEEN 80®).

As used herein, a "polymer" is a molecule formed by the covalent bonding of two or more monomers, where the monomers are not amino acid residues. Examples of polymers include, but are not limited to, polyethylene glycol, polypropylene glycol and copolymers (e.g. PLURONICS™, PF68 and the like), polyethylene glycol (PEG) such as PEG 400 and PEG 8000.

The terms "ion-exchange" and "ion-exchange chromatography" refer to a chromatographic process in which a target solute (such as a protein) in a mixture is reacted with a charged compound bound (eg, covalently attached) to an ion-exchange solid phase material, wherein the target solute interacts non-specifically with a charged compound stronger or weaker than with dissolved impurities or contaminants in the mixture. The contaminant solute in the mixture elutes from the ion exchange column faster or slower than the target solute, or binds or does not bind to the resin as opposed to the target solute. "Ion exchange chromatography" specifically includes cation exchange, anion exchange, and combination chromatography.

The phrase "ion exchange material" refers to a solid phase that is charged negatively (ie cation exchange resin) or positively (ie anion exchange resin). The charge may be due to the attachment to the solid phase of one or more charged ligands, for example, by covalent bonding. Alternatively or additionally, the charge may be a property of the solid phase itself (eg, as in the case of silica, which carries an overall negative charge).

"Solid phase" means a non-aqueous matrix to which one or more charged ligands can be attached. The solid phase may be a purification column, a dispersed particulate phase, a membrane or filter, and the like. Examples of solid phase forming materials include polysaccharides (such as agarose and cellulose) and other mechanically stable matrices such as silica (such as controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles, and derivatives thereof.

"Cation exchange resin" refers to a solid phase which is negatively charged and which thus carries free cations to exchange with cations in aqueous solution passing through the solid phase. The negatively charged ligand attached to the solid phase to form the cation exchange resin may, for example, be a carboxylate or a sulfonate. Commercially available cation exchange resins include, but are not limited to, carboxymethyl cellulose, sulfopropyl (SP) immobilized on agarose (e.g. SP-SEPHAROSE FAST FLOW (or SP-SEPHAROSE HIGH PERFORMANCE)) and sulfonyl immobilized on agarose (e.g. S- SEPHAROSE FAST FLOW), and POROS®HS.

"Combined ion exchange resins" refers to a solid phase that is modified by the covalent attachment of cationic, anionic and hydrophobic moieties. Combined ion exchange resins are also referred to as "multimodal ion exchange resins". Combined ion exchange resins are commercially available and include, for example, BAKERBOND ABX containing weak cation exchange groups, a low concentration of anion exchange groups, and hydrophobic ligands attached to a silica gel solid phase support. Additional examples of combined ion exchange resins include, but are not limited to, CAPTO ^™ Adhere resin, QMA resin, CAPTO ^™ MMC resin, MEP HyperCel resin, HEA HyperCel resin, PPA HyperCel resin, or ChromaSorb or Sartobind STIC membrane. In some embodiments, the composite material is a CAPTO ^™ Adhere resin.

The term "anion exchange resin" is used herein to refer to a solid phase that is positively charged, for example, has one or more positively charged ligands, such as quaternary amino groups, attached to it. Commercially available anion exchange resins include DEAE cellulose, QAE SEPHADEX, FAST Q SEPHAROSE ^TM and Q SEPHAROSE ^TM FAST FLOW.

A "buffer" is a solution that is resistant to changes in pH due to the conjugate action of its acid-base components. Depending on parameters such as the desired pH, various buffers can be used, which are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). In some cases, the buffer may have a pH in the range of about 2 to about 9, alternatively, from about 3 to about 8, alternatively, from about 4 to about 7, alternatively, from about 5 to about 7. Non-limiting examples of buffers that will control pH in this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate and ammonium buffer, as well as combinations thereof.

The term "hydrophobic interaction chromatography" or "HIC" is used herein to refer to a chromatographic process in which molecules are separated based on their hydrophobicity. Examples of resins that can be used for HIC include, but are not limited to, phenyl-, butyl-, octyl-sepharose (SEPHAROSE), BUTYL-SEPHAROSE® 4 Fast Flow, PHENYL SEPHAROSE ^TM High Performance, PHENYL SEPHAROSE ^TM 6 Fast Flow (low sub) and PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub). Typically, sample molecules are applied to a HIC column in high salt buffer. The salt in the buffer interacts with water molecules and reduces the solvation of molecules in solution, which leads to the exposure of hydrophobic regions of the sample molecules and, consequently, to their adsorption on the HIC column. The more hydrophobic the molecule, the lower the salt concentration needed to ensure binding. Typically, a decreasing salt gradient is used to elute samples from the column. Due to the decrease in ionic strength, the hydrophilic regions of the molecules begin to be exposed to a greater extent, and the molecules elute from the column in order of increasing hydrophobicity. Sample elution can also be accomplished by adding mild organic modifiers or detergents to the elution buffer.

"Application buffer" is a buffer that is used to apply a composition comprising a target polypeptide molecule and one or more impurities to an ion exchange resin. The loading buffer has a conductivity and/or pH that allows the target polypeptide molecule (and usually one or more contaminants) to bind to the ion exchange resin, or a conductivity and/or pH that allows the target protein to pass through the column while how impurities bind to the resin.

An "intermediate buffer" is used to elute one or more impurities from the ion exchange resin prior to eluting the target polypeptide molecule. The intermediate buffer has a conductivity and/or pH that allows the elution of one or more impurities from the ion exchange resin, but does not result in the elution of a significant amount of the target polypeptide.

As used herein, the term "wash buffer" refers to a buffer used to wash or equilibrate an ion exchange resin prior to eluting the target polypeptide molecule. In some cases, for convenience, wash buffer and application buffer may be the same, but this is not required.

An "elution buffer" is used to elute the target polypeptide from the solid phase. The elution buffer has a conductivity and/or pH that allows the target polypeptide to be eluted from the ion exchange resin.

A "regeneration buffer" can be used to regenerate the ion exchange resin so that it can be reused. The regenerating buffer has the conductivity and/or pH required to remove substantially all impurities and the target polypeptide from the ion exchange resin.

The term "conductivity" refers to the ability of an aqueous solution to conduct an electrical current between two electrodes. The solution conducts current through ion transport. Therefore, as the number of ions present in an aqueous solution increases, the conductivity of the solution will increase. The unit of conductivity is millisiemens per centimeter (mS/cm), and conductivity can be measured using a commercially available conductivity meter, such as the conductivity meter from Orion. The conductivity of a solution can be changed by changing the concentration of the ions it contains. For example, to achieve the desired conductivity, the concentration of the buffering agent and/or the concentration of the salt (eg, NaCl or KCl) in the solution can be changed.

The "pI" or "isoelectric point" of a polypeptide refers to the pH value at which the positive charge of the polypeptide is balanced by its negative charge. pI can be calculated as the net charge of the amino acid or sialic acid residues of the attached polypeptide carbohydrates, or can be determined using isoelectric focusing.

"Binding" of a molecule to an ion exchange material means the contact of the molecule with the ion exchange material under appropriate conditions (pH/conductivity) which results in the molecule being reversibly immobilized in or on the ion exchange material by ionic interactions between the molecule and the charged group or charged groups. ion exchange material.

"Washing" the ion exchange material means passing the appropriate buffer through the ion exchange material.

To "elute" a molecule (e.g., a polypeptide or contaminants) from an ion exchange material means to remove the molecule from the ion exchange material by changing the ionic strength of the buffer surrounding the ion exchange material such that the buffer competes with the molecule for charged binding sites on the ion exchange material.

"Ultrafiltration" is a form of membrane filtration in which hydrostatic pressure forces liquid through a semi-permeable membrane. Suspended solids and high molecular weight solutes remain above the membrane while water and low molecular weight solutes pass through the membrane. In some examples, ultrafiltration membranes have pore sizes ranging from 1 to 100 nm. The terms "ultrafiltration membrane" and "ultrafiltration filter" are used interchangeably.

"Diafiltration" is a method that involves the use of ultrafiltration membranes to remove salts or other dissolved microcomponents from a solution. Small molecules are separated from the solution due to the fact that large molecules remain in the retentate. This process uses selective semi-permeable (porous) membrane filters (filtration membranes) to separate components of solutions and suspensions based on their molecular size.

As used herein, the term "filtrate" refers to that part of the sample that passes through the filtration membrane.

As used herein, the term "retentate" refers to that portion of the sample that is substantially retained by the filtration membrane.

The term "pharmaceutical dosage form" refers to a preparation that is in such a form as to provide effective biological activity of the active ingredient contained therein, and which does not contain additional components that are unacceptably toxic to the subject to whom the dosage form will be administered.

A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical dosage form, other than the active ingredient, that is non-toxic to the subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, the term "treatment" refers to clinical intervention in an attempt to reverse the natural course of a condition in an individual being treated, and may be done for prophylaxis or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or temporary relief of the disease state, and remission or improvement in prognosis. In some embodiments, antibodies are used to delay the development of a disease or slow the progression of a disease.

Reference herein to a value or parameter with the term "about" includes (and describes) variations that are characteristic of that value or parameter as such. For example, an indication of "about X" includes an indication of "X".

ANTI-IL13 ANTIBODIES

In some embodiments, isolated and purified antibodies are provided that bind to IL-13. Examples of anti-IL13 antibodies are known and include, for example, lebrikizumab, IMA-026, IMA-638 (also called anrukinzumab, INN number 910649-32-0; QAX-576), tralokinumab (also called CAT-354, CAS number 1044515- 88-9); AER-001, ABT-308 (also called 13C5.5 humanized antibody), but are not limited to. Examples of such anti-IL13 antibodies and other IL13 inhibitors are disclosed, for example, in WO 2005/062967, WO2008/086395, WO2006/085938, US 7,615,213, US 7,501,121, WO2007/036745, WO2010/073119, WO2007/04547. In one embodiment, the anti-IL13 antibody is a humanized IgG4 antibody. In one embodiment, the anti-IL13 antibody is lebrikizumab. In one embodiment, the anti-IL13 antibody comprises three heavy chain CDRs, CDR-H1 (SEQ ID NO.: 1), CDR-H2 (SEQ ID NO.: 2) and CDR-H3 (SEQ ID NO.: 3 ). In one embodiment, the anti-IL13 antibody comprises three light chain CDRs, CDR-L1 (SEQ ID NO.: 4), CDR-L2 (SEQ ID NO.: 5) and CDR-L3 (SEQ ID NO.: 6 ). In one embodiment, the anti-IL13 antibody comprises three heavy chain CDRs and three light chain CDRs, CDR-H1 (SEQ ID NO.: 1), CDR-H2 (SEQ ID NO.: 2), CDR-H3 (SEQ ID NO.: 3), CDR-L1 (SEQ ID NO.: 4), CDR-L2 (SEQ ID NO.: 5) and CDR-L3 (SEQ ID NO.: 6). In one embodiment, the anti-IL13 antibody comprises a heavy chain variable region, VH, having an amino acid sequence selected from SEQ ID NO. 7 and 8. In one embodiment, the anti-IL13 antibody comprises a light chain variable region, VL, having the amino acid sequence of SEQ ID NO.: 9. In one embodiment, the anti-IL13 antibody comprises a heavy chain variable region, VH, having an amino acid sequence selected from SEQ ID NO. 7 and 8 and a light chain variable region, VL, having the amino acid sequence of SEQ ID NO.: 9. In one embodiment, the anti-IL13 antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO.: 10, or SEQ ID NO. .: 11, or SEQ ID NO.: 12, or SEQ ID NO.: 13. In one embodiment, the anti-IL13 antibody comprises a light chain having the amino acid sequence of SEQ ID NO.: 14. In one embodiment, The anti-IL13 antibody comprises a heavy chain having an amino acid sequence selected from SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, and SEQ ID NO.: 13, and a light chain having the amino acid sequence of SEQ ID NO.: 14.

In another aspect, the anti-IL-13 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO.: 8. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, or 99% sequence identity, contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-IL-13 antibody comprising such a sequence retains the ability to bind to human IL-13. In some embodiments, SEQ ID NO.: 8 has been replaced, altered by insertion and/or deletion of just 1 to 10 amino acids. In some embodiments, substitutions, insertions, or deletions are present in regions outside of the CDR (ie, in the FR). Optionally, the anti-IL13 antibody comprises the VH sequence of SEQ ID NO.: 8, including post-translational modifications of this sequence.

In another aspect, an anti-IL-13 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO.: 9. In some embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, or 99% sequence identity, contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an anti-IL-13 antibody comprising such a sequence retains the ability to bind to human IL-13. In some embodiments, SEQ ID NO.: 9 has been replaced, altered by insertion and/or deletion of just 1 to 10 amino acids. In some embodiments, substitutions, insertions, or deletions are present in regions outside of the CDR (ie, in the FR). Optionally, the anti-IL13 antibody comprises the VL sequence of SEQ ID NO.: 9, including post-translational modifications of this sequence.

In yet another embodiment, the anti-IL-13 antibody comprises a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100% sequence identity with the amino acid sequence of SEQ ID NO.: 9, and a VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO.: 8.

The table below lists the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions of lebrikizumab, as well as the VH, VL, heavy chain, and light chain sequences. As indicated in Table 1 below, the VH and the heavy chain may include an N-terminal glutamine, and the heavy chain may further include a C-terminal lysine. As is well known in the art, N-terminal glutamine residues can form pyroglutamate and C-terminal lysine residues can be removed during the production process.

Table 1. Amino acid sequences of anti-IL13 antibody (lebrikizumab).

CDR-H1
(SEQ ID NO.:1) Ala Tyr Ser Val Asn CDR-H2
(SEQ ID NO.:2) Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys Ser CDR-H3
(SEQ ID NO.:3) Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn CDR-L1
(SEQ ID NO.:4) Arg Ala Ser Lys Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His CDR-L2
(SEQ ID NO.:5) Leu Ala Ser Asn Leu Glu Ser CDR-L3
(SEQ ID NO.:6) Gln Gln Asn Asn Glu Asp Pro Arg Thr vh
(SEQ ID NO.:7) Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr Ser Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Gly Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser Leu Val Thr Val Ser Ser vh
(SEQ ID NO.:8) Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln
Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr
Ser Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
Ala Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys
Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala
Gly Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser
Leu Val Thr Val Ser Ser VL
(SEQ ID NO.:9) Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ser Val Ser Leu Gly
Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Asp Ser Tyr
Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg H chain
(SEQ ID NO.:10) VTLRESGPA LVKPTQTLTL TCTVSGFSLS AYSVNWIRQP PGKALEWLAM
IWGDGKIVYN SALKSRLTIS KDTSKNQVVL TMTNMDPVDT ATYYCAGDGY
YPYAMDNWGQ GSLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDY
FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYT
CNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLM
ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV
VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP
PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG
SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLG H chain
(SEQ ID NO.:11) QVTLRESGPA LVKPTQTLTL TCTVSGFSLS AYSVNWIRQP PGKALEWLAM
IWGDGKIVYN SALKSRLTIS KDTSKNQVVL TMTNMDPVDT ATYYCAGDGY
YPYAMDNWGQ GSLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDY
FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYT
CNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLM
ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV
VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP
PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG
SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLG H chain
(SEQ ID NO.:12) VTLRESGPA LVKPTQTLTL TCTVSGFSLS AYSVNWIRQP PGKALEWLAM
IWGDGKIVYN SALKSRLTIS KDTSKNQVVL TMTNMDPVDT ATYYCAGDGY
YPYAMDNWGQ GSLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDY
FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYT
CNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLM
ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV
VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP
PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG
SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLGK H chain
(SEQ ID NO.:13) QVTLRESGPA LVKPTQTLTL TCTVSGFSLS AYSVNWIRQP PGKALEWLAM
IWGDGKIVYN SALKSRLTIS KDTSKNQVVL TMTNMDPVDT ATYYCAGDGY
YPYAMDNWGQ GSLVTVSSAS TKGPSVFPLA PCSRSTSEST AALGCLVKDY
FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYT
CNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL FPPKPKDTLM
ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV
VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP
PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG
SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLGK L chain
(SEQ ID NO.: 14) DIVMTQSPDS LSVSLGERAT INCRASKSVD SYGNSFMHWY QQKPGQPPKL
LIYLASNLES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQQNNEDPR
TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV
QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV
THQGLSSPVT KSFNRGEC

OTHER RECOMBINANT POLYPEPTIDES

Recombinant polypeptides produced in CHO cells can be purified according to the methods described herein to remove or reduce the level of hamster PLBL2 so that only a residual or undetectable amount of hamster PLBL2 remains. Such polypeptides include, without limitation, growth factors, cytokines, immunoglobulins, antibodies, peptibodies, and the like.

Some examples of antibodies include anti-Abeta, anti-IL17A/F, and anti-CMV. Examples of anti-Abeta antibodies and methods for producing such antibodies have been previously described, for example, in WO2008011348, WO2007068429, WO2001062801 and WO2004071408. Examples of anti-IL17 A/F antibodies and methods for producing such antibodies have been described previously, for example, in WO 2009136286 and US patent number 8,715,669. Examples of anti-CMV antibodies, including anti-CMV-MSL, and methods for making such antibodies have been described previously, for example, in WO 2012047732.

Examples of polypeptides include mammalian proteins such as, for example, CD4; integrins and their subunits such as beta7; growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid-stimulating hormone; lipoproteins; ct-1-antitrypsin; A-chain of insulin; B-chain of insulin; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor and von Willebrand factor; anti-clotting factors such as protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator such as urokinase or tissue plasminogen activator (t-PA, eg Activase®, TNKase®, Retevase®); bombasine; thrombin; tumor necrosis factor-α and -β; enkephalinase; RANTES (chemokine expressed and secreted by T cells normally upon activation); human macrophage inflammatory protein (MIP-I-a); serum albumin, such as human serum albumin; Müllerian inhibitory substance; mouse gonadotropin-associated peptide; DNase; inhibin; activin; vascular endothelial growth factor (VEGF); IgE, receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotropin-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6) or a nerve growth factor such as NGF- β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I); proteins that bind insulin-like growth factor; other CD proteins such as CD3, CD8, CD19 and CD20; erythropoietin (EPO); thrombopoietin (TPO); osteoinductive factors; immunotoxins; bone morphogenetic protein (BMP); an interferon such as interferon-α, -β or -γ; colony stimulating factors (CSF) such as M-CSF, GM-CSF and G-CSF; interleukins (IL), e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL -24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33 and so on; superoxide dismutase; T cell receptors; surface membrane proteins; Complement Acceleration Factor (DAF); a viral antigen, such as, for example, part of the envelope of HIV; transport proteins; homing receptors; addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c, CD18; integrin subunits such as alpha4, alphaE, beta7; cell adhesion molecules such as ICAM, VLA-4 and VCAM; a tumor-associated antigen such as HER1 receptor (EGFR), HER2, HER3 or HER4; Apo2L/TRAIL and fragments of any of the above polypeptides; as well as immunoadhesins and antibodies that bind to them; and biologically active fragments or variants of the proteins listed above.

Additional examples of polypeptides include brain polypeptides including, but not limited to, beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolyprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine repeat-rich kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), receptor neutrophins p75 (p75NTR), P-selectin and caspase 6, and fragments of the above polypeptides; as well as immunoadhesins and antibodies that bind to them; and biologically active fragments or variants of the proteins listed above.

Other examples of polypeptides include therapeutic antibodies and immunoadhesins, including but not limited to antibodies, including antibody fragments, to one or more of the following antigens: HER1 (EGFR), HER2 (e.g., trastuzumab, pertuzumab), HER3, HER4, VEGF ( eg, bevacizumab, ranibizumab), MET (eg, onartuzumab), CD20 (eg, rituximab, obinutuzumab, ocrelizumab), CD22, CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, VCAM, IL-17A, and/or F , IgE (eg, omalizumab), DR5, CD40, Apo2L/TRAIL, EGFL7 (eg, parsatuzumab), NRP1, integrin beta7 (eg, etrolizumab), IL-13 (eg, lebrikizumab), Abeta (eg, crenezumab , gantenerumab), P-selectin (eg, inclacumab), IL-6R (eg, tocilizumab), IFNα (eg, rontalizumab), M1prime (eg, quilizumab) , mitogen-activated protein kinase (MAPK), OX40L, TSLP, factor D (eg lampalizumab) and receptors such as IL-9 receptor, IL-5 receptor, IL-4 receptor alpha chain, alpha 1 IL-13 receptor chain and alpha 2 IL-13 receptor chain, OX40, TSLP-R, IL-7R alpha (co-receptor for TSLP), IL17RB (receptor for IL-25), ST2 (receptor for IL-33), CCR3, CCR4, CRTH2, Fc epsilon RI and Fc epsilon RII/CD23 (receptors for IgE). Other examples of antibodies include, but are not limited to, antibodies selected from anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibodies, anti-CA15-3 antibodies, anti-CA19-9 antibodies, anti-c-erbB-2 antibodies, anti-P-glycoprotein antibodies, anti-CEA antibodies, anti-retinoblastoma protein antibodies, anti-ras kobelka antibodies, anti-Lewis X antibodies, anti-Ki-67 antibodies, anti-PCNA antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD5 antibodies, anti-CD7 antibodies, anti-CD8 antibodies, anti-CD9/p24 antibodies, anti-CD10 antibodies, anti-CD11c antibodies, anti-CD13 antibodies, anti-CD14 antibodies, anti-CD15 antibodies, anti-CD19 antibodies, anti-CD23 antibodies, anti-CD30 antibodies, anti-CD31 antibodies, anti-CD33 antibodies, anti-CD34 antibodies, anti-CD35 antibodies, anti-CD38 antibodies, anti-CD41 antibodies, anti-LCA/CD45 antibodies, anti-CD45RO antibodies, anti-CD45RA antibodies, anti-CD39 antibodies, anti-CD100 antibodies, anti-CD95/Fas antibodies , anti-CD99 antibodies, anti-CD106 antibodies, anti-ubiquitin antibodies, anti-CD71 antibodies, anti-c-myc antibodies, antibodies against cytokeratins, antibodies against vimentins, antibodies against HPV proteins, antibodies against kappa light chains, antibodies against lambda light chain antibodies, anti-melanosome antibodies, anti-prostate-specific antigen antibodies, anti-S-100 antibodies, anti-tau antibodies, anti-fibrin antibodies, anti-keratin antibodies, and anti-Tn antigen antibodies.

SOME CLEANING METHODS

The protein to be purified using the methods described herein is usually obtained by recombinant methods. Methods for producing recombinant proteins are described, for example, in US Pat. Nos. 5,534,615 and 4,816,567, specifically incorporated herein by reference. In some embodiments, the target protein is produced by a CHO cell (see, for example, WO 94/11026). Examples of proteins, including anti-IL13 monoclonal antibodies (anti-IL13 MAb), which can be purified using the processes described herein, have been described above.

When using recombinant methods, the protein can be produced intracellularly, in the periplasmic space, or can be secreted directly into the environment. If the protein is produced intracellularly, in the first step, particulate debris, either host cell fragments or lysed fragments, is removed by centrifugation or ultrafiltration. When the protein is secreted into the medium, the recombinant host cells can be separated from the culture medium, for example, using tangential flow filtration.

Protein A immobilized on the solid phase is used to purify the anti-IL13 MAb preparation. In some embodiments, the solid phase is a column having a glass, silica, agarose, or polystyrene surface to immobilize Protein A. In some embodiments, the solid phase is a controlled pore glass column or a silicic acid column. In some cases, the column is coated with a reagent such as glycerol to prevent non-specific binding to the column. The PROSEP A™ column, commercially available from Bioprocessing Limited, is an example of a protein A controlled pore glass column that is coated with glycerol. Other examples of columns provided herein include a POROS® 50 ATM column (polystyrene), or a SEPHAROSE FAST FLOW™ rProtein A column (agarose), or a MABSELECT SURE ^TM column (agarose) available from GE Healthcare Life Sciences (agarose).

The solid phase for protein A chromatography is balanced with an appropriate buffer. For example, the balancing buffer may be 25 mM Tris, 25 mM NaCl, pH 7.70 + 0.20.

A preparation derived from recombinant host cells and containing impurities and/or contaminants is applied to the equilibrated solid phase using an application buffer, which may be the same as the equilibrating buffer. As the drug containing impurities/contaminants passes through the solid phase, the protein is adsorbed onto the immobilized protein A, and other impurities/contaminants (such as Chinese Hamster Ovary Cell Proteins, CHOP if the protein is produced in the CHO cell) can non-specifically bind to the solid phase.

The next step, carried out sequentially, removes impurities/contaminants bound to the solid phase, antibody and/or protein A by washing the solid phase in an intermediate washing step. After application, the solid phase may be equilibrated with an equilibration buffer prior to an intermediate washing step.

The intermediate wash buffer may include a salt and optionally an additional compound such as (a) a detergent (eg polysorbate, eg polysorbate 20 or polysorbate 80); (b) a solvent (such as hexylene glycol); and (c) a polymer (such as polyethylene glycol [PEG]).

The salt used may be selected depending on the target protein. Examples of salts include but are not limited to sodium acetate, sodium citrate and potassium phosphate.

The amount of salt and additional compound (if present) in the composition should be such that their total amount is sufficient to elute the impurity(s)/contaminant(s) without substantially removing the protein of interest. Examples of salt concentrations in such wash buffers are from about 0.1 M to about 2 M, or from about 0.2 M to about 0.6 M. Detergent concentrations used are from about 0.01% to about 5%, or from about 0.1% to 1%, or about 0.5%, for example when the detergent is polysorbate. Examples of solvent concentrations are from about 1% to 40%, or from about 5 to about 25%. When the additional compound is a polymer (eg, PEG 400 or PEG 8000), its concentration may be, for example, from about 1% to about 20%, or from about 5% to about 15%.

The pH of the intermediate wash buffer is typically from about 4 to about 8, or from about 4.5 to about 5.5, or about 5.0. In one embodiment, the pH is 7.00 + 0.10.

After the intermediate washing step described above, the target protein is removed from the column. This is usually achieved by using a suitable elution buffer. The protein, for example, can be eluted from the column using an elution buffer having a low pH (acidic conditions), for example, in the range of about 2 to about 5, or in the range of about 2.5 to about 3.5. Examples of eluting buffers for this purpose include citrate or acetate buffers.

The eluted protein preparation may be subjected to additional purification steps either before or after the Protein A chromatography step. Examples of additional purification steps include hydroxyapatite chromatography; dialysis; affinity chromatography using an antibody to capture the protein; hydrophobic interaction chromatography (HIC); ammonium sulfate precipitation; anion exchange or cation exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on silica; chromatofocusing; ultrafiltration-diafiltration (UFDF) and gel filtration. In the examples herein, a Protein A chromatography step is followed by anion exchange (e.g., Q-Sepharose-Fast Flow) or multimodal (e.g., combined) ion exchange chromatography (e.g., CAPTO ^™ Adhere) and HIC steps (e.g., PHENYL SEPHAROSE ^TM 6 fast flow - high sub).

The protein thus isolated may be formulated with a pharmaceutically acceptable carrier and used for various diagnostic, therapeutic or other uses known for such molecules.

In some embodiments of any of the methods described herein, the chromatographic material is an ion exchange chromatographic material; for example, material for anion exchange chromatography. In some embodiments, the anion exchange chromatography material is a solid phase that is positively charged and contains free anions to exchange with the anions in the aqueous solution passing through the solid phase. In some embodiments of any of the methods described herein, the anion exchange material may be a membrane, a monolithic material, or a resin. In one embodiment, the anion exchange material may be a resin. In some embodiments, the anion exchange material may include a primary amine, a secondary amine, a tertiary amine, or a quaternary ammonium ion, a polyamine functional group, or a diethylaminoethyl functional group. In some of the embodiments above, the anion exchange chromatography material is an anion exchange chromatography column. In some of the embodiments above, the anion exchange chromatography material is an anion exchange chromatography membrane.

In some embodiments of any of the methods described herein, the ion exchange material may be a conventional chromatographic material or a convection chromatographic material. Convection chromatographic materials include, for example, perfusion materials (eg, poly(styrene-divinylbenzene) resin) and diffuse materials (eg, cross-linked agarose resin). In some embodiments, the poly(styrene-divinylbenzene) resin may be a POROS® resin. In some embodiments, the cross-linked agarose resin may be a sulfopropyl sepharose Fast Flow ("SPSFF") resin. The convection chromatographic material may be a membrane (eg, polyethersulfone) or a monolithic material (eg, a cross-linked polymer). The polyethersulfone membrane may be a Mustang membrane. The cross-linked polymer monolithic material may be a cross-linked poly(glycidyl methacrylate-co-ethylene dimethacrylate).

Examples of anion exchange materials include, but are not limited to, POROS® HQ 50, POROS® PI 50, POROS® D, Mustang Q, Q SEPHAROSE ^TM FF and DEAE Sepharose.

In some aspects, the chromatographic material is a hydrophobic interaction chromatography material. Hydrophobic Interaction Chromatography (HIC) is a liquid chromatography technique in which biomolecules are separated based on their hydrophobicity. Examples of HIC chromatographic materials include Toyopearl hexyl 650, Toyopearl butyl 650, Toyopearl phenyl 650, Toyopearl ether 650, Source, Resource, Sepharose Hi-Trap, octylsepharose, PHENYL SEPHAROSE ^TM high performance, PHENYL SEPHAROSE ^TM 6 fast flow (low sub) and PHENYL SEPHAROSE ^TM 6 fast flow (high sub), but not limited to. In some of the embodiments above, the HIC chromatography material is a HIC chromatography column. In some of the embodiments above, the HIC chromatographic material is a HIC chromatographic membrane.

In some aspects, the chromatographic material is an affinity chromatography material. Examples of affinity chromatography materials include, but are not limited to, Protein A or Protein G derivatized chromatographic materials. Examples of affinity chromatography materials include, but are not limited to, Prosep-VA, Prosep-VA Ultra Plus, fast flow Protein A Sepharose, Tyopearl Protein A, MAbSelect, MABSELECT SURE ^™ and MABSELECT SURE ^™ LX. In some of the embodiments above, the affinity chromatography material is an affinity chromatography column. In some of the embodiments above, the affinity chromatography material is an affinity chromatography membrane.

Depending on such factors as, for example, the desired pH of the buffer, the desired conductivity of the buffer, the characteristics of the target protein and the method of purification, various buffers can be used. In some embodiments of any of the methods described herein, the methods include the use of a buffer. The buffer may be a loading buffer, an equilibration buffer, or a wash buffer. In some embodiments, one or more of the buffers selected from application buffer, equilibration buffer, and/or wash buffer are the same. In some embodiments, the application buffer, equilibration buffer, and/or wash buffer are different buffers. In some embodiments of any of the methods described herein, the buffer includes a salt. The application buffer may include sodium chloride, sodium acetate, or a mixture thereof. In some embodiments, the application buffer is sodium chloride buffer. In some embodiments, the application buffer is sodium acetate buffer.

In this document, the term application is the application of the composition to the chromatographic material. The buffer for application is a buffer used to apply the composition, including the target product, to the chromatographic material. The chromatographic material may be equilibrated with an equilibration buffer prior to application of the composition to be purified. In some examples, a wash buffer is used after applying the composition to the chromatographic material and before elution of the target polypeptide from the solid phase. However, some target product, such as a polypeptide, may be removed from the chromatographic material with a wash buffer (eg, in breakthrough collection mode).

As used herein, the term elution is the removal of a product, such as a polypeptide, from a chromatographic material. An elution buffer is a buffer used to elute a polypeptide or other target product from a chromatographic material. In many cases, the physical characteristics of the elution buffer differ from the physical characteristics of the loading buffer. For example, the conductivity of the elution buffer may differ from that of the loading buffer, or the pH of the elution buffer may differ from the pH of the loading buffer. In some embodiments, the conductivity of the elution buffer is lower than the conductivity of the loading buffer. In some embodiments, the conductivity of the elution buffer is higher than the conductivity of the loading buffer. In some embodiments, the pH of the elution buffer is lower than the pH of the loading buffer. In some embodiments, the pH of the elution buffer is higher than the pH of the loading buffer. In some embodiments, the conductivity and pH of the elution buffer is different from the conductivity and pH of the loading buffer. The elution buffer may have any combination of higher or lower conductivity and higher or lower pH.

Conductivity refers to the ability of an aqueous solution to conduct an electrical current between two electrodes. The solution conducts current through ion transport. Therefore, as the number of ions present in an aqueous solution increases, the conductivity of the solution will increase. The basic unit of measure for conductivity is the siemens (or mo), mo (mS/cm) and conductivity can be measured using a commercially available conductivity meter such as from Orion. Since electrolytic conductivity is the ability of ions in a solution to conduct an electric current, the conductivity of a solution can be changed by changing the concentration of ions in the solution. For example, the concentration of the buffering agent and/or the concentration of the salt (eg, sodium chloride, sodium acetate, or sodium chloride) in the solution can be changed to obtain the desired conductivity. Preferably, the salt concentration is varied in different buffers to obtain the desired conductivity.

In some embodiments of any of the methods described herein, the flow rate is less than about 50 column volumes/hour, 40 column volumes/hour, or 30 column volumes/hour. The flow rate may be between about 5 CV/hr and 50 CV/hr, 10 CV/hr and 40 CV/hr, or 18 CV/hr and 36 CV/hr. In some embodiments, the flow rate is about 9 CV/hr, 18 CV/hr, 25 CV/hr, 30 CV/hr, 36 CV/hr, or 40 CV/hr. In some embodiments of any of the methods described herein, the flow rate is less than about 100 cm/hr, 75 cm/hr, or 50 cm/hr. The flow rate can be between about 25 cm/h and 150 cm/h, 25 cm/h and 100 cm/h, 50 cm/h and 100 cm/h or 65 cm/h and 85 cm/h or 50 cm/h and 250 cm/h or 100 cm/h and 250 cm/h or 150 cm/h and 250 cm/h.

The layer height is the height of the chromatographic material used. In some embodiments of any of the methods described herein, the layer height is greater than about 3 cm, 10 cm, or 15 cm. The layer height may be between 3 cm and 35 cm, 5 cm and 15 cm, 3 cm and 10 cm, or 5 cm and 8 cm. In some embodiments, the layer height is about 3 cm, 5 cm, 10 cm, or 15 cm. In some embodiments, the layer height is determined based on the amount of polypeptide or contaminants in the substance applied to the column.

In some embodiments, the chromatography is carried out on a carrier column that is greater than about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml. ml, 25 ml, 30 ml, 40 ml, 50 ml, 75 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 800 ml, 900 ml, 1 l, 2 l, 3 l, 4 l, 5 l, 6 l, 7 l, 8 l, 9 l, 10 l, 25 l, 50 l, 100 l, 200 l, 400 l or 450 l.

In some embodiments, fractions are collected during chromatography. In some embodiments, the volume of fractions collected is greater than about 0.01 column volumes, 0.02 column volumes, 0.03 column volumes, 0.04 column volumes, 0.05 column volumes, 0.06 column volumes, 0. 07 column volumes, 0.08 column volumes, 0.09 column volumes, 0.1 column volumes, 0.2 column volumes, 0.3 column volumes, 0.4 column volumes, 0.5 column volumes, 0.6 column volumes column, 0.7 column volumes, 0.8 column volumes, 0.9 column volumes, 1.0 column volumes, 2.0 column volumes, 3.0 column volumes, 4.0 column volumes, 5.0 column volumes . In some embodiments, fractions containing a product, such as a polypeptide, are pooled. In some embodiments, the fractions containing the polypeptide from the applied fractions and from the eluted fractions are pooled. The amount of polypeptide in the fraction can be determined by a person skilled in the art; for example, the amount of polypeptide in a fraction can be determined using UV spectroscopy. In some embodiments, fractions containing a detectable polypeptide fragment are pooled.

In some embodiments of any of the methods described herein, at least one impurity or contaminant is one or more substances selected from host cell substances, such as CHOP; leached protein A; nucleic acid; variant, fragment, aggregate or derivative of the target polypeptide; another polypeptide; endotoxin; viral contaminant; culture medium component, gentamicin and the like. In some examples, the impurity or contaminant may be a host cell protein (HCP), e.g., a bacterial cell such as an E. coli cell, an insect cell, a prokaryotic cell, a eukaryotic cell, a yeast cell, a mammalian cell, an avian cell, a cell mushroom, but not limited to.

Host cell proteins (HCPs) are proteins from cells in which the polypeptide is produced. For example, CHOPs are proteins from host cells, ie. Chinese hamster ovary cell proteins. The number of CHOP can be measured using enzyme-linked immunosorbent assay ("ELISA") or mass spectrometric analysis. In some embodiments of any of the methods described herein, the amount of HCP (e.g., CHOP) is reduced by more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. The amount of HCP can be reduced by approximately between 10% and 99%, 30% and 95%, 30% and 99%, 50% and 95%, 50% and 99%, 75% and 99% or 85% and 99% . In some embodiments, the amount of HCP is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 98%. In some embodiments, the reduction is determined by comparing the amount of HCP in the composition obtained after the purification step(s) with the amount of HCP in the composition prior to the purification step(s).

In some embodiments of any of the methods described herein, the methods further comprise obtaining a purified polypeptide. In some embodiments, a purified polypeptide is obtained after any of the purification steps described herein. The chromatography step may be anion exchange chromatography, HIC, or protein A chromatography. In some embodiments, the first chromatography step is protein A chromatography, followed by anion exchange or multimodal ion exchange chromatography, and then HIC.

In some embodiments, the polypeptide is further purified after chromatography by viral filtration. Viral filtration consists in removing viral contaminants from the polypeptide fraction to be purified. Examples of viral filtration include ultrafiltration and microfiltration. In some embodiments, the polypeptide is purified using a parvo filter.

In some embodiments, the polypeptide is concentrated after chromatography. Examples of concentration methods are known in the art and include, but are not limited to, ultrafiltration and diafiltration.

In some embodiments of any of the methods described herein, the methods further comprise combining the polypeptide purified according to the purification methods with a pharmaceutically acceptable carrier.

Monoclonal antibodies

In some embodiments, the antibodies purified according to the methods of the present invention are monoclonal antibodies. Monoclonal antibodies are derived from a population of substantially homogeneous antibodies, ie. the individual antibodies in a population are identical and/or bind to the same epitope, except for possible variations that may arise during the production of a monoclonal antibody, but such variations are usually present in minor amounts. Thus, the modifier "monoclonal" indicates the nature of the antibody, which is not a mixture of single or polyclonal antibodies.

For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be made by recombinant DNA methods (US Pat. No. 4,816,567).

In the hybridoma method, a mouse or other suitable host animal such as a hamster is immunized as described herein to activate lymphocytes that produce or can produce antibodies that will specifically bind to the polypeptide used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form a hybridoma cell (Goding, Monoclonal antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus obtained are planted and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma culture media will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

In some embodiments, the myeloma cells are cells that fuse efficiently, maintain a high level of stable antibody production by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. In some embodiments, the myeloma cell line includes mouse myeloma cell lines, such as myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors, available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and cells SP-2 or X63-Ag8-653, available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal antibody Production Techniques and Applications pp. 51-63 ( Marcel Dekker, Inc., New York, 1987)).

The culture medium in which the hybridoma cells grow is used to evaluate the production of monoclonal antibodies directed against the antigen. In some embodiments, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme immunoassay (ELISA).

The binding affinity of a monoclonal antibody can be determined, for example, using the Scatchard assay, as described in Munson et al., Anal. Biochem. 107:220 (1980).

Once hybridoma cells have been identified that produce antibodies of the desired specificity, affinity, and/or activity, clones can be subcloned by serial dilution and grown by standard methods (Goding, Monoclonal antibodies: Principles and Practice pp. 59-103 (Academic Press, 1986)) . Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascitic tumors in animals.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascitic fluid or serum using conventional immunoglobulin purification methods such as, for example, protein A sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

DNA encoding monoclonal antibodies can be easily isolated and sequenced by conventional methods (eg, using oligonucleotide probes that can specifically bind to the genes encoding heavy and light chains of mouse antibodies). In some embodiments, hybridoma cells serve as the source of such DNA. Once isolated, the DNA can be introduced into expression vectors that are further transfected into host cells such as E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not produce an immunoglobulin polypeptide to synthesize monoclonal antibodies in recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol. 5:256-262 (1993) and Plückthun, Immunol. Revs. 130:151-188 (1992).

In another embodiment, antibodies or antibody fragments can be isolated from an antibody phage library generated using the methods described in McCafferty et al., Nature 348:552-554 (1990). In Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991) describes the isolation of mouse and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology 10:779-783 (1992)), as well as using combinatorial infection and in vivo recombination as strategies to generate very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Thus, these methods represent viable alternatives to traditional hybridoma methods for the production and isolation of monoclonal antibodies.

The DNA can be further modified, for example, by replacing the murine heavy and light chain constant domain encoding sequences with homologous human sequences (US Pat. No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA 81:6851 (1984)) or by covalently linking all or some of the non-immunoglobulin polypeptide coding sequences to an immunoglobulin coding sequence.

Typically, such non-immunoglobulin polypeptides replace the constant domains of an antibody, or they replace the variable domains of an antigen-binding active site of an antibody to create a chimeric bivalent antibody comprising one antigen-binding active site with specificity for an antigen and another antigen-binding active site with specificity for the other. antigen.

In some embodiments of any of the methods described herein, the antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody is an IgG monoclonal antibody.

Humanized antibodies

In some embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies have been described in the art. In some embodiments, the humanized antibody contains one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "imported" residues, and are typically taken from the "import" variable domain. Humanization can generally be carried out according to the method proposed by Winter et al. (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al. al., Science 239:1534-1536 (1988)), by replacing the corresponding human antibody sequences with hypervariable region sequences. Accordingly, such "humanized" antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which a sequence substantially smaller than that of an intact human variable domain has been replaced with a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced with residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy chain, which are used to obtain humanized antibodies, is very important to reduce their antigenicity. According to the so-called "best fit" method, the variable domain sequence of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence closest to the rodent sequence is then adopted as the human framework region (FR) for the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions. The same framework region can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993 )).

In addition, it is important that humanized antibodies retain high antigen affinity and other favorable biological properties. To achieve this goal, in some embodiments of the methods, humanized antibodies are generated through a process of analyzing the original sequences and various putative humanized products using three-dimensional models of the original and humanized sequences. Three-dimensional models of immunoglobulins are widely available and known to those skilled in the art. Computer programs are available that generate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. The study of these images allows us to analyze the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. analyze residues that affect the ability of a candidate immunoglobulin to bind to its antigen. Therefore, FR residues can be selected from the recipient and import sequences and combined in such a way as to obtain the desired characteristics of the antibody, such as increased affinity for the target antigen(s). In general, hypervariable region residues are directly and most significantly involved in antigen binding and affect antigen binding.

human antibodies

In some embodiments, the antibody is a human antibody. As an alternative to humanization, human antibodies can be created. For example, it is now possible to obtain transgenic animals (eg mice) that are capable of producing the full repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the junction region (J _H ) of the antibody heavy chain gene in chimeric and germline mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such mice with germline mutations will result in the production of human antibodies upon antigen challenge. See, for example, Jakobovits et al., Proc. Natl. Acad. sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Year in Immuno. 7:33 (1993); and US Patent Numbers 5,591,669; 5,589,369; and 5,545,807.

Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable (V) gene repertoires from immunized donors (McCafferty et al., Nature 348:552-553 (1990)). According to this method, genes of the V domain of an antibody are cloned into the frame of a major or minor coat polypeptide gene of a filamentous bacteriophage, such as M13 or fd, and it is expressed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a copy of the single-stranded DNA representing the genome of the phage, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody having those properties. Thus, the phage mimics some properties of B cells. Phage display can be implemented in different formats; a review of phage display is provided, for example, in Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a broad array of anti-oxazolone antibodies from a small random combinatorial V gene library derived from the spleens of immunized mice. A repertoire of V genes from immunized human donors can be generated and antibodies to a diverse set of antigens (including self antigens) can be isolated essentially following the methods described in Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

Human antibodies can also be generated in vitro using activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

Antibody fragments

In some embodiments, the antibody is an antibody fragment. Various methods have been developed to obtain antibody fragments. Traditionally, these fragments have been prepared by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However, these fragments can now be generated directly from recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries, as discussed above. Alternatively, Fab'-SH fragments can be directly obtained from E. coli and chemically linked to form F(ab') ₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). In accordance with another approach, fragments of F(ab') ₂ can be isolated directly from the culture of recombinant host cells. Other methods for obtaining antibody fragments will be apparent to the skilled practitioner. In other embodiments, the target antibody is a single chain Fv fragment (scFv). See WO 93/16185; US patent number 5,571,894; and US patent number 5,587,458. An antibody fragment can also be a "linear antibody" such as described in US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

In some embodiments, the antibody fragments described herein are provided. In some embodiments, the antibody fragment is an antigen binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, an F(ab') ₂ fragment, scFv, Fv, and a diabody.

Chimeric polypeptides

The polypeptides described herein may be modified in certain ways to form chimeric molecules comprising a polypeptide fused to another heterologous polypeptide or amino acid sequence. In some embodiments, the chimeric molecule includes a polypeptide fused to a tag polypeptide that provides an epitope to which the anti-tag antibody selectively binds. An epitope tag is usually located at the amino or carboxyl terminus of a polypeptide. The presence of such epitope-tagged forms of the polypeptide can be detected using an antibody against the tagged polypeptide. In addition, the presence of an epitope tag allows the polypeptide to be easily purified by affinity purification using an anti-tag antibody or other type of affinity template that binds to the epitope tag.

Other

Another type of covalent modification of a polypeptide involves linking the polypeptide to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The polypeptide may also be encapsulated in microcapsules, for example, by coacervation or interfacial polymerization techniques (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such methods are disclosed in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, A.R., Ed., (1990).

OBTAINING POLYPEPTIDES

The polypeptides used in the purification methods described herein can be obtained using methods well known in the art, including recombinant methods. The following sections provide guidance on these methods.

Polynucleotides

The terms "polynucleotide" or "nucleic acid", used interchangeably herein, refer to polymers of nucleotides of any length and include DNA and RNA.

The polynucleotides encoding the polypeptides may be obtained from any source, including, but not limited to, cDNA libraries obtained from tissue believed to contain the mRNA of the polypeptide and express it at a detectable level. Accordingly, polynucleotides encoding a polypeptide can be readily obtained from a cDNA library generated from human tissue. The gene encoding the polypeptide can also be obtained from a genomic library or by known synthetic methods (eg, automated nucleic acid synthesis).

For example, a polynucleotide may encode a full length immunoglobulin chain molecule, such as a light chain or a heavy chain. The complete heavy chain includes not only the heavy chain variable region (V _H ), but also the heavy chain constant region (CH ₎ , which will typically include three constant domains: C _H 1, C _H 2 and C _H 3; and "hinged" area. In some situations, having a constant region is desirable.

Other polypeptides that may be encoded by a polynucleotide include antigen-binding antibody fragments such as single domain antibodies ("dAb"), Fv, scFv, Fab', F(ab') ₂ and "minibodies". Minibodies are (typically) bivalent antibody fragments from which the C _H 1 and C _K or C _L domain has been removed. Because miniantibodies are smaller than conventional antibodies, they should have better tissue penetration in clinical/diagnostic applications, but, being bivalent, they should retain higher binding affinity than monovalent antibody fragments such as dAbs. Accordingly, unless the context dictates otherwise, the term "antibody" as used herein includes not only full-length antibody molecules, but also antigen-binding fragments of antibodies belonging to the types discussed above. Preferably, each framework present in the encoded polypeptide will include at least one amino acid substitution compared to the corresponding human acceptor framework. Thus, for example, the framework regions may include a total of three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen amino acid substitutions compared to the acceptor framework.

Suitable polynucleotides described herein may be isolated and/or purified. In some embodiments, the polynucleotides are isolated polynucleotides.

The term "isolated polynucleotide" is intended to indicate that the molecule is removed from its normal or natural environment, or separated from its normal or natural environment, or has been produced in such a way that it is not in its normal or natural environment. In some embodiments, the polynucleotides are purified polynucleotides. The term purified indicates that at least some of the contaminating molecules or substances have been removed.

Suitable polynucleotides are substantially purified such that the corresponding polynucleotides are the dominant (ie, most represented) polynucleotides in the composition.

Expression of polynucleotides

The description below relates primarily to the production of polypeptides by culturing cells transformed or transfected with a vector containing polynucleotides encoding a polypeptide. Of course, it is contemplated that alternative methods well known in the art can be used to obtain polypeptides. For example, the corresponding amino acid sequence, or portion thereof, can be obtained by direct peptide synthesis using solid phase synthesis methods (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis W. H. Freeman Co., San Francisco, Calif. (1969); Merrifield , J. Am. Chem. Soc. 85:2149-2154 (1963)). Protein synthesis in vitro can be carried out using manual or automated methods. Automated synthesis can be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) following the manufacturer's instructions. The various parts of a polypeptide can be chemically synthesized separately and then combined using chemical or enzymatic methods to obtain the desired polypeptide.

The polynucleotides as described herein are placed in an expression vector(s) to effect production of the polypeptides. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences include, but are not limited to, promoters (eg, naturally associated promoters or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.

A polynucleotide is "operably linked" when it is placed into a functional dependency of another polynucleotide sequence. For example, nucleic acids for a presequence or secretory leader are operably linked to nucleic acids for a polypeptide if it is expressed as a precursor protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence if it is located in such a way that it facilitates translation. Generally, "operably linked" means that the linked nucleic acid sequences are contiguous and, in the case of a secretory leader, contiguous and in the same reading frame. However, enhancers do not have to border on an operably related sequence. Linking is accomplished by ligation at suitable restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with common practice.

In the case of antibodies, the light and heavy chains can be cloned into the same expression vector or into different expression vectors. The nucleic acid segments encoding the immunoglobulin chains are operably linked to control sequences in the expression vector(s) that allow expression of the immunoglobulin polypeptides.

Vectors containing polynucleotide sequences (e.g., variable heavy and/or variable light chain encoding sequences, and optionally expression control sequences) can be introduced into the host cell by well known methods, which differ depending on the type of host cell. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolisting, or viral transfection can be used for other host cells. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. To create transgenic animals, transgenes can be microinjected into fertilized oocytes, or the transgene can be introduced into the genome of embryonic stem cells, and the nuclei of such cells can be transferred into non-nucleated oocytes.

Vectors

The term "vector" includes expression vectors, transform vectors and transfer vectors.

The term "expression vector" means a construct capable of in vivo or in vitro expression.

The term "transforming vector" means a construct that can be transferred from one host to another host, which may be of the same species or different species. If the construct can be transferred from one species to another, for example from an Escherichia coli plasmid to another bacterium, such as a bacterium of the genus Bacillus, then the transforming vector is sometimes referred to as a "transfer vector". It may even be a construct that can be transferred from an E. coli plasmid to an Agrobacterium and a plant.

The vectors can be transformed into a suitable host cell, as described below, to allow expression of the polypeptide. Various vectors are public. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The corresponding nucleotide sequence can be inserted into the vector using various techniques. In general, the DNA is inserted into the appropriate restriction endonuclease cleavage site (restriction endonuclease cleavage sites) using methods known in the art. Suitable vectors containing one or more of these components are constructed using standard ligation techniques known to those skilled in the art.

The vectors may be, for example, a plasmid, a virus or a phage vector containing an origin of replication, optionally a promoter for the expression of said polynucleotide, and optionally a promoter regulator. The vectors may contain one or more selectable marker genes, which are well known in the art.

These expression vectors typically replicate in host organisms either as episomes or as an integral part of chromosomal DNA.

Host cells

The host cell may be, for example, a bacterial cell, a yeast or other fungal cell, an insect cell, a plant cell, or a mammalian cell.

To obtain a polypeptide, a genetically engineered transgenic multicellular host organism can be used. The organism may be, for example, a transgenic mammal (eg, a transgenic strain of goats or mice).

Suitable prokaryotes include, but are not limited to, eubacteria such as Gram-negative or Gram-positive organisms, for example Enterobacteriaceae such as E. coli. Various strains of E. coli are publicly available, for example E. coli strain K12 MM294 (ATCC 31,446); E. coli X1776 (ATCC 31.537); E. coli strain W3110 (ATCC 27.325) and K5 772 (ATCC 53.635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella such as Salmonella typhimurium, Serratia such as Serratia marcescans and Shigella as well as Bacilli such as B subtilis and B. licheniformis (eg B. licheniformis 41P), Pseudomonas such as P. aeruginosa and Streptomyces. These examples are illustrative and are not intended to be limiting. The W3110 strain is a particularly preferred host strain or parental host strain as it is commonly used as a host strain for the preparation of a recombinant polynucleotide product. Preferably, the host cell secretes a minimal amount of proteolytic enzymes. For example, the W3110 strain may be modified to contain a genetic mutation in genes encoding host cell endogenous polypeptides, for example, including E. coli strain W3110 1A2 having the full tonA genotype; strain E. coli W3110 9E4 with full tonA ptr3 genotype; E. coli strain W3110 27C7 (ATCC 55,244) having the full genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan'; strain E. coli W3110 37D6 with full genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan'; E. coli strain W3110 40B4, which is a 37D6 strain that lost resistance to kanamycin due to a degP deletion mutation; and an E. coli strain having a mutant periplasmic protease. Alternatively, in vitro cloning methods, such as PCR or other nucleic acid polymerase reactions, may be used.

Expression vectors can be introduced into these prokaryotic host cells, which will typically contain expression control sequences compatible with the host cell (eg, origin of replication). In addition, any number of different well-known promoters may be present, such as the lactose operon promoter system, the tryptophan operon (trp) promoter system, the beta-lactamase operon promoter system, or the promoter system from phage lambda. Promoters will typically control expression, optionally from an operator sequence, and will contain a ribosome binding site sequence and the like for the initiation and completion of transcription and translation.

Eukaryotic microorganisms can be used for expression. Eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable hosts for cloning or expression of vectors encoding the polypeptide. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Other host organisms include Schizosaccharomyces pombe; Kluyveromyces such as, for example, K. lactis (MW98-8C, CBS683, CBS4574), K. fragilis (ATCC 12.424), K. bulgaricus (ATCC 16.045), K. wickeramii (ATCC 24.178), K. waltii (ATCC 56.500 ), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; yarrowia (EP 402,226); Pichia pastoris; Candida Trichoderma reesia; Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium and Aspergillus, such as A. nidulans and A. niger. Methylotrophic yeasts are suitable for the purposes of this document and include, but are not limited to, yeasts that can grow on methanol selected from the genera Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. Saccharomyces are preferred as yeast hosts using suitable vectors containing expression control sequences (eg, promoters), origin of replication, termination sequences, and the like, as appropriate. Common promoters include the promoter of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from the genes for alcohol dehydrogenase, isocytochrome C, and maltose and galactose utilization enzymes.

In addition to microorganisms, mammalian tissue culture cells can also be used to express and produce polypeptides as described herein, and in some cases this is preferred (see Winnacker, From Genes to Clones VCH Publishers, N.Y., N.Y. (1987) For some embodiments, eukaryotic cells may be preferred because a number of suitable host cell lines capable of secreting heterologous polypeptides (e.g., intact immunoglobulins) have been developed in the art and include CHO cell lines, various Cos cell lines, HeLa, preferably myeloma cell lines, or transformed B cells, or hybridomas In some embodiments, the mammalian host cell is a CHO cell.

In some embodiments, the host cell is a vertebrate host cell. Examples of useful mammalian host cell lines are CV1 monkey kidney cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cells (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese Hamster Ovary Cells/-DHFR (CHO or CHO-DP-12 line); mouse Sertoli cells; monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); gray rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatoma cells (Hep G2).

DOSAGE FORMS AND METHODS FOR OBTAINING THE DOSAGE FORM

Also provided herein are dosage forms and methods for creating dosage forms comprising polypeptides (eg, antibodies) purified by the methods described herein. For example, the purified polypeptide may be combined with a pharmaceutically acceptable carrier.

Dosage forms containing a polypeptide, in some embodiments, can be prepared for storage by mixing a polypeptide having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ), in the form of lyophilized dosage forms or aqueous solutions.

As used herein, "carriers" include pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to a cell or mammal, in which they are administered at the dosages and concentrations used. Often the physiologically acceptable carrier is an aqueous solution with a pH buffer.

Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the doses and concentrations used and include buffers such as phosphate, citrate, histidine buffer and buffers based on other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In some embodiments, the polypeptide in a dosage form containing the polypeptide retains functional activity.

Dosage forms used for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.

Dosage forms herein may further contain more than one active compound if required by the particular indication being treated, preferably they may contain active compounds with complementary activities that do not adversely affect each other. For example, in addition to the polypeptide, the dosage form may optionally include an additional polypeptide (eg, antibody) in one dosage form. Alternatively or additionally, the composition may further comprise a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an antihormonal agent, and/or a cardioprotective agent. Such molecules may be present in combination in amounts effective for the intended purposes.

Examples of dosage forms of anti-IL13 antibodies described herein are provided in international published patent application number WO 2013/066866.

PRODUCTS

Polypeptides purified by the methods described herein and/or dosage forms containing polypeptides purified by the methods described herein may be contained in a product. The article of manufacture may include a container containing the polypeptide and/or a dosage form containing the polypeptide. In some embodiments, the article of manufacture includes: (a) a container containing a composition comprising a polypeptide and/or a dosage form containing a polypeptide described herein in a container; and (b) a package leaflet with instructions for administering the dosage form to a subject.

The product includes a container and a label or leaflet placed in or otherwise associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. Containers can be made from different materials such as glass or plastic. The container contains the dosage form and may have a sterile inlet (for example, the container may be a bag of intravenous solution or a vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a polypeptide. The label or package insert indicates that the composition is being used by the subject, and provides specific guidance regarding dosages and dosage intervals for the intended polypeptide and any other drug. The product may additionally include other material that is desirable from a commercial and consumer point of view, including other buffers, diluents, filters, needles and syringes. In some embodiments, the container is a syringe. In some embodiments, the syringe is part of an injection device. In some embodiments, the injection device is an auto-injector.

"Packlet" is used to refer to the instructions normally included in commercial packaging of therapeutic products, which contains information about indications, uses, dosages, administration, contraindications, other therapeutic products with which the packaged product is compatible, and/or warnings regarding use such therapeutic products.

Examples of articles containing dosage forms of anti-IL13 antibodies described herein are provided in international published application number WO 2013/066866.

Additional details of the invention are illustrated by the following non-limiting Examples. Disclosures of all references in the description are expressly incorporated herein by reference.

EXAMPLES

As used in the Examples below and throughout this document, "PLB2", "PLBL2", and "PLBD2" are interchangeable and refer to "phospholipase B-like enzyme 2" or, as a synonym, to "phospholipase B-domain-like enzyme 2" .

EXAMPLE 1 General Methods

Materials and methods for all Examples were as follows unless otherwise noted in the Example.

Stock concentrated solutions of MAb

The stock MAb stock solutions in all examples were selected from industrial, experimental, or low-volume cell culture batches obtained from Genentech (South San Francisco, CA, USA). After the cell culture fermentation period, the cells were separated and, in some cases, the clarified liquid (harvested culture liquid, HCCF) was purified by protein A chromatography and one or more additional chromatography and filtration steps as indicated in the Examples below.

MAb quantification

Antibody concentration was determined by absorbance at 280 and 320 nm using a UV-visible spectrophotometer (8453 model G1103A; Agilent Technologies; Santa Clara, CA, USA) or NanoDrop 1000 model ND-1000 (Thermo Fisher Scientific; Waltham, MA, USA). The concentration of non-antibody substances (ie, impurities) was too low to noticeably affect the absorption in the UV region. If necessary, the samples were diluted with an appropriate non-reactive diluent in the range of 0.1-1.0 optical units. Sample preparation and UV measurements were performed in duplicate and the average value was recorded. MAb uptake ratios ranged from 1.42 to 1.645/mg.ml.cm.

Total CHO Host Cell Protein Quantification (CHOP)

ELISA was used to quantify the levels of a total host cell protein called CHOP. The ELISA used to detect CHO proteins in foods was based on the ELISA in sandwich assay format. The affinity-purified anti-CHOP polyclonal antibody was coated on a 96-well microtiter plate. Standards, controls and samples were applied in duplicate in two separate wells. CHOPs, if present in the sample, bind to the antibody-coated surface of the plate (polyclonal anti-CHOP). After the incubation step, an anti-CHOP polyclonal antibody conjugated with horseradish peroxidase (HRP) was added to the plate. After the final wash step, CHOP was quantified by adding tetramethylbenzidine (TMB) solution, also available as SUREBLUE RESERVE ^™ from KPL, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, cat. no. 53-00-03), which upon reaction with the HRP enzyme gives a colorimetric signal. Optical density (OD) at 450 nm was measured in each well. A five-parameter point-to-point curve fitting program (SOFTMAX® Pro, Molecular Devices, Sunnyvale, CA) was used to fit the standard curve and CHOP concentration in the samples was calculated from the fitted standard curve. The range of quantitation results for the ELISA for total CHOP was from 5 to 320 ng/mL. The CHOP concentration in ng/mL refers to the amount of CHOP in the sample using the CHOP standard as the calibrant. The CHOP ratio (in ng/mg or ppm) refers to the calculated ratio of CHOP concentration to product concentration and, in some cases, has been a reported value for the methods tested. The total CHOP ELISA can be used to quantify the total CHOP level in a sample, but is not suitable for quantifying the concentration of individual proteins.

ELISA assay with mouse anti-hamster PLBL2 monoclonal antibody

The production of mouse anti-hamster PLBL2 monoclonal antibodies and the development of an ELISA assay for detecting and quantifying PLBL2 in a preparation of recombinant polypeptides using such antibodies is described in US Provisional Applications 61/877,503 and 61/991,228. Briefly, the assay was performed as described below.

Mouse monoclonal antibody 19C10 was coated on a half-well 96-well microtiter plate at 0.5 μg/ml in carbonate buffer (0.05M sodium carbonate, pH 9.6) overnight at 2-8°C. After coating the surface of the plate, it was blocked with blocking buffer (0.15M NaCl, 0.1M sodium phosphate, 0.1% fish gelatin, 0.05% polysorbate 20, 0.05% Proclin® 300 [Sigma-Aldrich]; also called solution for dilution) to prevent non-specific protein binding. Standards, controls, and samples were diluted in dilution solution (0.15M NaCl, 0.1M sodium phosphate, 0.1% fish gelatin, 0.05% polysorbate 20, 0.05% Proclin® 300 [Sigma-Aldrich]) and then applied in duplicate in separate wells and incubated for 2 h at room temperature (22-27°C). PLBL2, if present in the sample, will bind to the antibody (also referred to herein as "captured" by antibody) covering the surface of the plate. After the incubation step above, unbound substances were washed with wash buffer (0.05% polysorbate 20/PBS [Corning cellgro catalog number 99-717-CM]) and mouse biotin-conjugated anti-PLBL2 monoclonal antibody 15G11 was diluted dilution solution to a concentration of 0.03125 µg/ml and added to the wells of the microtiter plate.

Conjugation with biotin was carried out as follows. The biotinylation kit was obtained from Pierce Thermo Scientific, (P/N 20217, E-Z Link NHS-Biotin), and streptavidin-HRP (SA-HRP) was obtained from Jackson Immuno, catalog number 016-030-084. The instructions in the Pierce Kit were as follows. Briefly, IgG was dialyzed in PBS, pH 7.4, and biotin was added to the protein and mixed at room temperature for 1 h. The labeled antibody was further dialyzed against PBS, pH 7.4 to remove excess biotin, filtered, and protein concentration was determined by absorption at 280 nm (A280).

After a 2 hour incubation step with biotinylated 15G11 at room temperature, streptavidin HRP (1:200,000 dilution in dilution buffer) was added to the wells of the microtiter plate. After a final wash step with wash buffer (as described above), staining was obtained (for PLBL2 quantification) by adding a TMB solution (50 µl/well) (SUREBLUE RESERVE ^™ from KPL, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, catalog number 53-00-03) followed by incubation at room temperature for 10-20 minutes. Detection was performed by assessing the optical density (OD) at 450 nm in each well using Molecular Devices SpectraMax M5e. A four-parameter point-by-point curve fitting program (SoftMax Pro v5.2 rev C) was used to construct the standard curve, and PLBL2 concentrations in the samples were calculated over the linear range of the standard curve. Values in the linear range of the standard curve were used to calculate the nominal concentration of PLBL2 (ng/mg or ppm). The linear range was approximately between EC ₁₀ - EC ₈₅ or 1.5 - 40 ng/ml and varied slightly between plates. Values obtained for PLBL2 using this ELISA method were comparable to those obtained by other methods (eg, LC-MS/MS, polyclonal antibody ELISA for PLBL2, or total CHOP ELISA diluted to LOQ assay).

ELISA assay with rabbit anti-hamster PLBL2 polyclonal antibody

The production of rabbit anti-hamster PLBL2 polyclonal antibodies and the development of an ELISA assay to detect and quantify PLBL2 in a preparation of recombinant polypeptides using such antibodies is described in US Provisional Applications 61/877,503 and 61/991,228. Briefly, the assay was performed as described below.

Affinity purified rabbit polyclonal antibody was coated on a 96-well half-well microtiter plate at 0.5 μg/ml in carbonate buffer (0.05M sodium carbonate, pH 9.6) overnight at 2-8°C. After coating the surface of the plate, it was blocked with blocking buffer (0.15M NaCl, 0.1M sodium phosphate, 0.1% fish gelatin, 0.05% polysorbate 20, 0.05% Proclin® 300 [Sigma-Aldrich]; also called solution for dilution) to prevent non-specific protein binding. Standards, controls, and samples were diluted in dilution solution (0.15M NaCl, 0.1M sodium phosphate, 0.1% fish gelatin, 0.05% polysorbate 20, 0.05% Proclin® 300 [Sigma-Aldrich]) and then applied in duplicate in separate wells and incubated for 2 h at room temperature (22-27°C). PLBL2, if present in the sample, will bind to the antibody (also referred to herein as "captured" by antibody) covering the surface of the plate. After the incubation step above, unbound substances were washed with wash buffer (0.05% polysorbate 20/PBS [Corning cellgro catalog number 99-717-CM]), and affinity purified rabbit polyclonal antibody conjugated to horseradish peroxidase (HRP ), diluted with dilution solution to a concentration of 40 ng/ml and added to the wells of a microtiter plate.

HRP conjugation was carried out as follows. An HRP conjugation kit was obtained from Pierce Thermo Scientific, (P/N 31489, E-Z Link Plus Activated Peroxidase and Kit). The instructions in the Pierce Kit were as follows. Briefly, IgG was dialyzed in carbonate-bicarbonate buffer, pH 9.4, and EZ-Link Plus activated peroxidase was added to the protein and mixed at room temperature for 1 h. Next, sodium cyanoborohydride and quench buffer were added to stabilize the conjugation and quench the reaction. The labeled antibody was further dialyzed against PBS, pH 7.4, filtered and the protein concentration determined by absorbance at 280 nm (A280).

After a 2 hour incubation step with HRP-conjugated rabbit polyclonal antibody at room temperature, a final wash step was performed with wash buffer (described above). Thereafter, staining was obtained (for quantification of PLBL2) by adding a TMB solution (50 μl/well) (SUREBLUE RESERVE ^TM from KPL, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, cat. no. 53-00-03) followed by incubation at room temperature for 10-20 minutes. Detection was performed by assessing the optical density (OD) at 450 nm in each well using Molecular Devices SpectraMax M5e. A five-parameter point-by-point curve fitting program (SoftMax Pro v5.2 rev C) was used to construct the standard curve, and PLBL2 concentrations in the samples were calculated in the linear range of the standard curve. Values in the linear range of the standard curve were used to calculate the nominal concentration of PLBL2 (in ng/mg or ppm). The assay quantitation range was 0.5-50 ng/mL. Values obtained for PLBL2 using this ELISA method were comparable to those obtained by other methods (eg, by LC-MS/MS, polyclonal antibody ELISA for PLBL2, or total CHOP ELISA diluted to LOQ assay).

LC-MS/MS Analysis

A Waters Acquity H-Class Bio UPLC system and an AB Sciex TripleTOF 5600+ mass spectrometer were used to quantify PLBL2 by LC-MS/MS. Samples and calibration standards (recombinant PLBL2 added to a preparation of recombinant humanized monoclonal antibody derived from mouse NS0 cell line [NS0 cell line does not contain hamster PLBL2]) were reconstituted and digested with trypsin. A total of 40 µg of tryptic digest of the sample was injected into the UPLC system with a Waters BEH300 C18 column with a particle size of 1.7 μm. The peptides were eluted with a linear gradient of acetonitrile at a flow rate of 300 μl/min and a column temperature of 60°C.

Peptides eluted with UPLC were introduced into the mass spectrometer by electrospray ionization in the positive ion ionization mode. The ion source temperature was set to 400°C, the IonSpray voltage was 5500 V, and the declustering voltage was 76 V. An impact energy setting of 32 was used to fragment selected peptide ions. The mass spectrometer was set to operate in high resolution monitoring mode. multiple reactions (MRM ^HR ) for four specific peptides from PLBL2 and MRM transitions for their daughter ions. Precursor ions were sampled in a quadrupole mass spectrometer within a mass-to-charge ratio (m/z) ion sample window with a width of 1.2 amu. The daughter ions for each parent ion were separated from each other using a time-of-flight mass spectrometer and selected for quantification within a 0.025 amu wide window.

The concentration of PLBL2 in the samples was determined by measuring the specific signals of the four transitions, calibrated using standards with an accuracy of 2-500 ppm and linear approximation. Table 2 below lists the peptides from the PLBL2 protein as measured by LC-MS/MS.

Table 2. List of peptides from PLBL2 measured by LC-MS/MS

TripleTOF 5600+ scan cycle Scan # Scan Type Peptide Target child ion m/z parent ion m/z of the daughter ion 1 TOF MS N/A N/A N/A N/A 2 Daughter ion SVLLDAASGQLR
(SEQ ID NO: 31) +2y8 615.3461 817.4163 3 Daughter ion GLEDSYEGR
(SEQ ID NO: 32) +2y7 513.2304 855.3479 4 Daughter ion AFIPNGPSPGSR
(SEQ ID NO: 33) +2y9 600.3120 868.4272 5 Daughter ion VTSFSLAK
(SEQ ID NO: 34) +2y6 426.7449 652.3665

[0100] EXAMPLE 2 Improved purification process to reduce hamster PLBL2

[0101] In order to provide early stage clinical trials, a purification process for the anti-IL13 MAb (lebrikizumab) produced by CHO, referred to herein as the "Initial Process", was developed. The Initial Process uses the following chromatographic steps in the following order: protein A affinity chromatography (MABSELECT SURE ^TM ) followed by cation exchange chromatography (POROS® HS) and then ion exchange chromatography (Q SEPHAROSE ^TM Fast Flow). Additional virus inactivation and filtration steps and a final ultrafiltration-diafiltration (UFDF) step were included. The final product (drug) was formulated at 125 mg/ml in 20 mM histidine acetate with 6% sucrose, 0.03% polysorbate 20, pH 5.7.

Using the total CHOP ELISA assay (described in Example 1 above), for in-house intermediates and drug purified according to the Parent Process, we found an atypical dilution relationship characterized by a >20% coefficient of variation within normalized sample dilution series. This dilution dependence is illustrated by the data presented in Table 3, in which each subsequent 2-fold dilution of the anti-IL13 MAb product was associated with higher levels of CHOP (expressed in ppm) as shown by ELISA for total CHOP. Using sensitive analytical methods such as LC-MS/MS, we determined that single species CHOP or HCP was responsible for this atypical dilution relationship. In particular, we found that the cause of the dilution dependence observed in the total CHOP ELISA was an excess of antigen. Further investigation allowed us to determine that the single species HCP is hamster phospholipase B-like enzyme 2 (PLBL2). By diluting product samples to the assay limit of quantification (LOQ), we were able to evaluate the PLBL2 level in clinical batches of lebrikizumab purified using the Parent Process and determine that the level of PLBL2 present in them was greater than 300 ppm (300 ng/mg) and higher .

Table 3. Product Dilution and CHOP Levels

Dilution ratio Total CHOPs (ppm) 2 0.58 4 1 8 2 16 4 32 7 64 14 128 26 256 49 512 97 1024 147 2048 228 4096 314 8192 346

This observed level of impurities (> 300 ppm) CHOP single species is considered undesirable in MAb products intended for clinical and/or therapeutic use in humans, especially in the late stage of clinical trials and beyond. For example, such levels may be immunogenic when administered to human subjects as described in Example 3.

Accordingly, we have explored various modifications of the Origin Process, as summarized below. Based on the results of these studies, we have developed an improved purification process, detailed below and referred to herein as the "Improved Process". The improved process results in a purified anti-IL13 MAb product (lebrikizumab) containing substantially reduced levels of PLBL2.

Attempts to modify the purification process to reduce PLBL2 levels have included methods independent of the Initial Process, including precipitation, testing of various HCCF additives, additional column washes, hydrophobic interaction chromatography, and combination chromatography. One or more of the assays described in Example 1 were used to monitor the effectiveness of each of the test modifications in reducing total CHOP and/or PLBL2 levels. Various test modifications are described below.

Precipitation of CHOP by Caprylic Acid in HCCF and Pool after Protein A

Caprylic acid precipitation has been described previously, including its use in the industrial production of monoclonal antibodies (Wang et al., BioPharm International; Downstream Processing 2010, p4-10, Oct2009; Brodsky et al., Biotechnology and Bioengineering, 109(10):2589, 2012) for the selective precipitation of impurities from target proteins. Caprylic acid, also known as octanoic acid, is a saturated fatty acid with eight carbon atoms (formula CH ₃ (CH ₂ ) ₆ COOH). Studies were conducted with anti-IL13 MAbs to determine whether precipitation of harvested culture fluid (HCCF) or protein A pool with caprylic acid would result in a reduction in CHOP and/or a decrease in ELISA dilution dependence on total CHOP.

The initial source of anti-IL13 MAbs for these studies used HCCF and a post-Protein A pool from 1000 L of collected fluid. 1% (v/v) caprylic acid was added to the HCCF and various concentrations of caprylic acid (0% - 3% v/v) were added to the pools prepared using Protein A at pH 4.5 or pH 5.0. Samples were agitated for ≥ 5 hours at ambient temperature, filtered through a 0.2 µm filter, and diluted with a diluent designed for total CHOP ELISA for detection and quantification by total CHOP ELISA. The titer of anti-IL13 MAb in HCCF before and after treatment with caprylic acid was determined using HPLC titer analysis, which was carried out in accordance with standard methods known in the art.

Treatment of HCCF with 1% v/v caprylic acid resulted in a decrease in CHOP by approximately 5-fold and a yield of anti-IL13 MAb was 91%. When pools downstream of Protein A were treated with various concentrations of caprylic acid ranging from 0-3% v/v, we observed >20% reduction in yield at pH 5.0 and no reduction in yield at pH 4.5. When we assessed the total CHOP in these pools after caprylic acid treated protein A, we found a 2-fold to 3-fold reduction in CHOP (Fig. 1A and B). However, as also shown in FIG. 1A and B, a dilution dependency would still be observed for each condition tested, indicating that caprylic acid precipitation was not effective in addressing the dilution dependency problem seen in total CHOP ELISAs, and therefore also will not be effective in reducing PLBL2 levels in this product.

Additives to HCCF

Previous work by Sisodiya et al., Biotech J. 7:1233 (2012) showed that the addition of, for example, guanidine hydrochloride or sodium chloride to HCCF can lead to a decrease in CHOP levels in pools further purified on protein A. Since it was also Arginine has been shown to reduce CHOP levels when used to wash Protein A columns (Millipore Technical Bulletin, Lit. No. TB1024EN00, Rev. A, December, 2005; Millipore Technical Bulletin, Lit. No. 1026EN00, July, 2006, available at www.Millipore.com), we have included it as an additive to HCCF. Various salts, chaotropic agents and caprylic acid were added to the anti-IL13 MAb HCCF to evaluate the effectiveness of each in reducing the interaction of the product and CHOP in the process of binding the product to MABSELECT SURE ^TM (MSS) by protein A chromatography. The following additions to HCCF were tested. : 0.6M guanidine, 0.6M arginine, 0.6M NaCl, phosphate buffered saline and 1% caprylic acid.

Samples treated with each of the HCCF additives were subjected to protein A chromatography on MSS. Protein A pools were adjusted to pH 4.9 and further purified using a POROS® HS cation exchange chromatography step under the conditions specified in the Reference Process. Protein A pools and POROS® HS pools were diluted and analyzed by ELISA for total CHOPs. The pH-adjusted protein A pools were also tested for SEC-HPLC according to methods known in the art to evaluate % aggregates, % variants, and the like.

Yields on MABSELECT SURE ^™ were somewhat lower for experiments in which guanidine or arginine was added to HCCF. Of all HCCF supplements tested, guanidine and arginine were most effective in significantly reducing CHOP levels (see Table 4) and appeared to reduce dilution dependence for protein A pools (data not shown). Post-treatment of Protein A pools on POROS® HS, however, showed that the dilution dependence observed in the CHOP ELISA was maintained for the corresponding POROS® pools, as shown in FIG. 2. Accordingly, the data demonstrate that the addition of guanidine or arginine to HCCF is not effective in reducing PLBL2 levels in this product.

Table 4. Additives to HCCF and their effect on CHOP

Additive application pH Exit (%) Total CHOPs (ppm) Control (no additives) 7.4 101 3417 0.6M guanidine 7.6 90 892 0.6M arginine 7.1 88 1237 0.6M NaCl 7.7 99 2619 PBS 7.4 98 2773 1% caprylic acid 6 93 3173

Protein A column wash (MABSELECT SURE ^TM )

We observed that more dilution dependent CHOP eluted in earlier MABSELECT SURE ^™ (MSS) fractions containing product when run on Protein A chromatography. This suggested that an additional MSS wash step prior to elution could lead to a decrease in CHOP/PLBL2. The ability of several variants of MSS washes to reduce CHOP/PLBL2 levels in pools after protein A was tested. For this study, a pool of purified anti-IL13 MAb after UFDF was used as a coating material. The UFDF pool was diluted to 1.7 mg/mL (approximate titer in HCCF) and applied to MSS with 29 g/L resin. Various wash solutions have been tested, e.g. 0.5M arginine pH 8.5, 0.5M arginine pH 9.5 with and without 1% polysorbate 20, 0.5M TMAC pH 7.1, 25 mM MOPS pH 7.1 was compared with a high salt wash solution pH 7.0. The product was eluted under acidic conditions (pH 2.8) and fractions were pooled starting at 0.5 OD (A280) to a total volume of 2.4 column volumes. Each pool after adjusting the pH was diluted and analyzed using ELISA for total CHOP. The summary results are that none of the wash conditions resulted in a satisfactory reduction in CHOP/PLBL2 levels or ELISA dilution dependence on total CHOPs. Thus, it seems unlikely that we could find protein A column wash conditions that would be effective in reducing PLBL2 levels in this anti-IL13 MAb product, and we did not conduct further studies in this direction.

Cation Exchange Column Wash (POROS® HS)

Based on theoretical calculations using the amino acid sequence of the anti-IL13 MAb and the PLBL2 contaminant, we estimated that the pI of PLBL2 is approximately 6.0 and is similar to that of the anti-IL13 MAb (pI 6.1). We also calculated that the net charge difference between anti-IL13 MAb and PLBL2 would be significant at ≤ pH 4 and ≥ pH 10. Therefore, we tested different low pH wash solutions in the cation exchange chromatography step with POROS® HS in Stock process to determine if they would be effective in selectively reducing total CHOP and/or PLBL2 and dilution dependent. The following washes were tested at pH 4: (i) acetate gradient, 300 mM - 1,000 mM in 20 column volumes (CV); (ii) citrate gradient, 100 mM - 500 mM in 20 CV; (iii) washing step with 260 mM citrate; and (iv) an arginine gradient of up to 15 mS/cm (conductivity unit) in a volume of 20 CV.

The results showed that anti-IL13 MAb and CHOP failed to elute at pH 4 with an acetate gradient to the tested salt concentration of 1M. Increasing the amount of citrate or acetate caused the product to become insoluble and precipitate. All washes at pH 4 gave low yields in the POROS® HS step and none of the washes resulted in a significant reduction in CHOP dilution dependence. Accordingly, the use of low pH wash solutions to wash the cation exchange column was not effective in reducing PLBL2 levels in this product.

Hydroxyapatite resin and CAPTO ^TM Adhere resin

Macroporous resin based ceramic hydroxyapatite (CHT) type I, 40 μm, (BioRad) consists of calcium phosphate (Ca ₅ (PO ₄ ) ₃ OH) ₂ organized in repeating hexagonal structures. Two distinct binding sites are present on the resin: C sites with a set of 5 calcium ion doublets and P sites containing phosphate triplets containing -OH pairs. This resin has combined properties and has been shown to be able to separate difficult impurities such as aggregates (P. Gagnon, New Biotechnology 25(5):287 (2009)).

To determine the initial conditions for conducting CHT column chromatography, we performed a high-throughput robotic screening of CHT resin type I, 40 µm, testing it in the pH range of 6.5 - 8.0 and using various concentrations of sodium chloride and sodium phosphate for elution. Such high throughput automated screening has been previously described, for example, in Wensel et al., Biotechnol. Bioeng. 100:839 (2008). Samples after this screening were tested by ELISA for total CHOP.

CAPTO ^TM Adhere (GE Healthcare) is a combination resin that has both ionic and hydrophobic properties. The base matrix is solid agarose and the ligand is N-benzyl-N-methylethanolamine. The ability of this resin to reduce total CHOP and/or PLBL2 levels was evaluated first by high throughput screening and then under appropriate column conditions.

Initial studies to determine initial CAPTO ^™ Adhere column chromatography conditions were performed using a high-throughput robotic screen similar to that described above to test the binding of anti-IL13 MAb to CAPTO ^™ Adhere at two loading densities (5 g/L resin and 40 g/L resin). l resin). Also tested salts and pH ranges: 25 mm - 200 mm sodium acetate and pH 4.0 - 6.5. The coating material was a post-UFDF pool from the Initial Process containing approximately 200 ppm total CHOPs at LOQ as measured by total CHOP ELISA. Samples of unbound material (leakage) on CAPTO ^TM Adhere were diluted and analyzed using ELISA for total CHOP.

The results were as follows. In the case of CHT chromatography, none of the conditions tested substantially resulted in a decrease in total CHOP or PLBL2 levels or affected dilution dependence. In addition, the yields were low, and the purification of high molecular weight compounds was not achieved. For CAPTO ^™ Adhere chromatography, the yields were low and the analytes were shown to be ELISA dilution dependent on total CHOPs. Accordingly, the use of CHT and CAPTO ^™ Adhere resin was not further investigated as it was clear that we would be unlikely to find resin conditions that would be effective in reducing PLBL2 levels in this anti-IL13 MAb product.

Resins and membranes for hydrophobic interaction chromatography

We first tested a HIC membrane adsorber called Sartobind and manufactured by Sartorius. Sartobind is a regenerated cellulose base matrix with covalently bonded hydrophobic phenyl ligand groups.

The membrane tested was a 3 ml Sartobind HIC device (layer height 8 mm). We adjusted the potassium phosphate content of the POROS® HS pool from the Initial Process to 0.55 M, pH 7.0 and used a flow rate of 10 ml/min. The product was eluted with 0.55 M potassium phosphate pH 7.0 (collected as unbound fractions in a 3 ml fraction).

We observed that the anti-IL13 MAbs became opalescent and cloudy when conditioned to 0.55 M potassium phosphate, requiring an additional filtration step through a 0.2 µm filter. The results showed a reduction in total CHOPs, however, for the remaining CHOPs, there was still an ELISA dilution dependence on total CHOPs. No further study was conducted on the use of this membrane as it seemed unlikely to us that we would find conditions effective in reducing PLBL2 levels in this product.

Next, we used high-throughput screening to evaluate several different HIC resins. OCTYL-SEPHAROSE® Fast Flow (FF), BUTYL-SEPHAROSE® 4 Fast Flow, PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) and PHENYL SEPHAROSE ^TM 6 Fast Flow (low sub) resins were obtained from GE Healthcare. These four resins were selected because they covered a wide range of hydrophobicity (OCTYL-SEPHAROSE® Fast Flow is the least hydrophobic, followed by PHENYL SEPHAROSE ^TM 6 Fast Flow (low sub) and BUTYL-SEPHAROSE® 4 Fast Flow, the most hydrophobic being PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) We tested the effectiveness of several combinations of pH and salt concentrations in reducing PLBL2 levels in anti-IL13 MAb formulations The anti-IL13 MAb formulation used for the HIC Resin experiments was pooled after UFDF in the Initial Process The anti-IL13 MAb concentration was 180 mg/mL and the loading density was 40 mg antibody/mL resin We tested pH 5.5 (25 mM sodium acetate), pH 6.0 (25 mM MES), pH 7.0 ( 25 mM MOPS) and pH 8.0 (25 mM Tris) and sodium sulfate concentrations between 0 mM and 400 mM For each test condition, breakthrough samples were collected, diluted and tested by ELISA assay for total CHOPs.

The results are shown in FIG. 3A-D. As the salt concentration increased, we observed a decrease in total CHOPs in breakthrough for each resin. OCTYL-SEPHAROSE® Fast Flow Resin (FIG. 3A) showed the highest levels of total CHOPs, PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) resin reduced total CHOP levels to very low levels, even with lower salt concentrations (FIG. 3D), and PHENYL SEPHAROSE ^TM 6 Fast Flow (low sub) and BUTYL-SEPHAROSE® Fast Flow resins showed intermediate total CHOP levels. Interestingly, there was also a minimal effect of pH on CHOP removal with any of the resins except PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) under low salt conditions (FIG. 3D). For this resin, under low salt conditions, higher pH resulted in more CHOP in the breakthrough fraction (FIG. 3D). Based on these results, the use of PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) seemed promising, and it was chosen for further studies, which included the use of the column in either binding and elution mode or breakthrough collection mode.

For HIC application using PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) resin in binding and elution mode, the anti-IL13 MAb must be conditioned prior to salt loading to allow the antibody to bind to the resin. Increasing the salt concentration results in an increase in the dynamic binding capacity (mg anti-IL13/ml resin) for applying the product to the resin. But as the salt concentration in the product increased, we observed clouding and the formation of high molecular weight compounds (HMW), in particular in combination with low pH.

As mentioned above, PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) can also be used in breakthrough collection mode and this application also requires less pre-application salt conditioning. In terms of, for example, product quality and stability, a product with less haze and less HMW, and less salt required for conditioning prior to application, would be preferred. Accordingly, we ran the process of optimizing the application of PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) resin in breakthrough mode.

To optimize the process, we investigated numerous HIC column chromatography parameters, including loading concentration, loading pH, loading salt molarity, loading density on resin, bed height, flow rate, temperature, equilibration buffer pH, and molarity. For these experiments, we monitored total CHOP content by total CHOP ELISA as well as PLBL2 content by LC-MS/MS. Some examples of data are shown in Table 5. The data in Table 5 demonstrates that the HIC column used under these conditions in the breakthrough collection mode was effective in significantly reducing PLBL2 levels compared to high levels in the post-Protein A pool. PLBL2 levels after HIC were reduced by several hundred times compared to the levels in the pool after protein A.

Table 5. Total CHOP levels and PLBL2 levels under different HIC column usage conditions

Sample
(layer height, flow rate) Exit, % Total CHOP (ppm, as determined by ELISA at LOQ) PLBL2 (ppm, based on LC-MS/MS) Pool after protein A (applied to a HIC column) 3324 957 15 cm, 150 cm/h 88 43 4 25 cm, 150 cm/h 92 44 2 15 cm, 100 cm/h 90 67 5 25 cm, 100 cm/h 92 63 3 15 cm, 200 cm/h 93 62 6 25 cm, 200 cm/h 90 72 4 15 cm, 150 cm/h 54 76 2

Using LC-MS/MS analysis for PLBL2 and other typical product quality assays (e.g. SE-HPLC, CE-SDS, iCIEF) to select process parameters, evaluating product quality characteristics and PLBL2 reduction, we found that the following conditions are preferred for HIC column chromatography: equilibration and wash buffer: 50 mM sodium acetate, pH 5.0; application density of the target substance: 100 g/l, flow rate: 150 cm/h, 22°C ± 3°C. Some small variations in these conditions may also be desirable, such as 25°C ± 3°C or 27°C ± 3°C. Optical density (OD) was monitored by absorbance at 280 nm (A280) and a pool (ie, breakthrough) was collected between 0.5 OD and 1.5 OD or after washing with 8 column volumes.

As mentioned above, the Initial process was protein A affinity chromatography (MABSELECT SURE ^TM ), then cation exchange chromatography (POROS® HS) followed by anion exchange chromatography (Q SEPHAROSE ^TM Fast Flow). After developing a process to lower PLBL2 levels, as described above, we decided to modify the process accordingly. Accordingly, we explored the following options: adding a HIC column to the Initial Process (so the process involved using four columns), replacing the HIC column with either a CEX column or an AEX column, and finally we examined the column order. We found that a process using three columns, protein A affinity chromatography (MABSELECT SURE ^TM ), followed by anion exchange chromatography (Q SEPHAROSE ^TM Fast Flow) followed by HIC in breakthrough collection mode (PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub)) was the most convenient and most effective for reducing the level of PLBL2 in the final drug substance. This three-column process is detailed below.

The first stage of affinity chromatography was a process of binding and elution using resin MABSELECT SURE ^TM . After equilibrating the column (25 mM sodium chloride, 25 mM Tris, pH 7.7), HCCF was applied to the column and washed with equilibration buffer and high salt wash buffer pH 7.0. Anti-IL13 MAbs were eluted from the column under acidic conditions (pH 2.8).

The second stage of anion exchange chromatography was carried out in the mode of binding and elution using resin Q SEPHAROSE ^TM Fast Flow (QSFF). After column equilibration (50 mM Tris, pH 8.0), the pH of the anti-IL13 pool from the MABSELECT SURE ^TM column was adjusted to 8.05 and the pool was loaded onto the column. The column was washed (50 mM Tris, pH 8.0) and the anti-IL13 MAb was eluted from the column with 85 mM sodium chloride, 50 mM Tris, pH 8.0.

The third and final step of hydrophobic interaction chromatography was run in breakthrough mode using PHENYL SEPHAROSE ^TM 6 Fast Flow (High Sub) resin. After the column was equilibrated (50 mM sodium acetate pH 5.0), the anti-IL13 pool from the QSFF column was adjusted to pH 5.0 and the pool was loaded onto the column. The anti-IL13 MAb passed through the column and the column was further washed with equilibration buffer (50 mM sodium acetate pH 5.0). Anti-IL13 MAbs were collected based on the A280 measurement and fractions between 0.5 and 1.5 OD were pooled or fractions up to a maximum of 8 column volumes.

As with the Initial Process, additional virus inactivation and filtration steps and a final ultrafiltration-diafiltration (UFDF) step were included. The final product (drug) was formulated at 125 mg/ml in 20 mM histidine acetate with 6% sucrose, 0.03% polysorbate 20, pH 5.7.

Comparison of the Initial Process with the Improved Process in terms of total CHOP and PLBL2 measured by total CHOP ELISA and PLBL2 monoclonal ELISA, respectively, is shown in Tables 6 (Original Process) and 7 (Improved Process). The data in Table 6 clearly demonstrate that the Initial Process results in a purified product (Pool after UFDF) containing high levels of total CHOP (179, 310 and 189 ng/mg in three different experiments) and high levels of PLBL2 (242, 328 and 273 ng/mg in three different experiments), while the data in Table 7 clearly demonstrate that the Improved Process was quite effective in obtaining a purified product with significantly reduced total CHOP levels (1.1, <0.9, 2.8 and 3 .4 ng/mg in four different experiments) and significantly reduced levels of PLBL2 (0.21, 0.42, 0.35 and 0.24 ng/mg in four different experiments). In view of the data above, the data in Table 7 demonstrates that performing HIC column chromatography under the conditions described above was particularly effective in reducing total CHOP and PLBL2 levels in anti-IL13 MAb formulations.

Table 6. CHOP and PLBL2 levels at various steps in the Anti-IL13 MAb Initial Purification Process

Sample in the process of cleaning Total CHOP (ng/mg at LOQ, as determined by ELISA) PLBL2 (ng/mg, by ELISA) Experiment number 1 2 3 1 2 3 HCCF 620920 541072 608789 1895 3669 2535 Pool after protein A 2892 2855 3505 587 769 478 Pool after CEX 136 310 138 345 439 287 Pool after AEX 104 163 93 291 304 261 Pool after UFDF 179 310 189 242 328 273

Table 7. CHOP and PLBL2 levels at various stages of the Improved Anti-IL13 MAb Purification Process

Sample in the process of cleaning Total CHOP (ng/mg at LOQ, as determined by ELISA) PLBL2 (ng/mg, by ELISA) Experiment number 1 2 3 4 1 2 3 4 HCCF 332132 399157 540134 644549 4084 3770 3077 2986 Pool after protein A 2318 2768 3552 3797 1354 1995 1027 975 Pool after AEX 495 653 414 377 723 933 677 616 Pool after HIC < 2.1 < 1.9 5.0 7.7 < 0.6 < 0.6 < 0.6 < 0.6 Pool after UFDF 1.1 < 0.9 2.8 3.4 0.21 0.42 0.35 0.24

Finally, faced with the problem of non-linear dilution dependence associated with high levels of single species CHOP in purified anti-IL13 MAb preparations, we first identified single species CHOP as hamster PLBL2, an impurity that had not previously been described in recombinant protein preparations derived from CHO cells. Next, we determined purification conditions effective to reduce PLBL2 levels in anti-IL13 MAb preparations. Finally, we investigated these purification conditions as part of a general purification process improved over the original anti-IL13 MAb purification process. The Improved Process uses a HIC column in breakthrough collection mode to reduce PLBL2 levels in combination with an affinity chromatography step and an anion exchange chromatography step. We have shown that the Improved Process is a reliable and effective way to significantly reduce hamster PLBL2 levels in anti-IL13 MAb preparations. We have shown that the Improved Process reproducibly reduces PLBL2 levels by approximately 1000-fold compared to the Original Process. This reduction in PLBL2 levels is important in order to obtain a purified anti-IL13 MAb product suitable for therapeutic use by patients at the late stage of clinical trials and beyond.

Purification process to reduce hamster PLBL2 levels in anti-Abeta antibody preparations

Next, we decided to evaluate whether the purification methods described above, in particular methods using a HIC column in the final chromatography step, would also be effective in reducing PLBL2 levels in other antibody preparations. For this experiment, we chose an anti-Abeta antibody generated in CHO cells. Examples of anti-Abeta antibodies and methods for producing such antibodies have been described previously, for example in WO2008011348, WO2007068429, WO2001062801 and WO2004071408. In these specific experiments, we used an anti-Abeta antibody called crenezumab. As described for the anti-IL13 MAb, we tested different resins and buffers for the second column after the protein A affinity column, and we tested different buffers and chromatography conditions for the HIC column to select those resins, buffers and conditions that are optimal in terms of in terms of the quality and stability of the anti-Abeta product, and also in terms of hamster PLBL2 removal.

We found that a three-column process involving protein A affinity chromatography (MABSELECT SURE ^TM ) followed by combination resin (CAPTO ^TM Adhere) followed by HIC in breakthrough mode (PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub)) , was convenient and effective in reducing the level of PLBL2 in the final drug substance. This three-column process is detailed below.

The first affinity chromatography step was a coupling and elution process using MABSELECT SURE ^TM resin, similar to that described above for the anti-IL13 MAb.

The second stage of the combined chromatography was carried out in the collection of breakthrough using resin CAPTO ^TM Adhere. After column equilibration (20 mM MES, 150 mM sodium acetate, pH 6.25), the pH of the anti-Abeta pool from the MABSELECT SURE ^TM column was adjusted to 6.25 and the pool was loaded onto the column. Fractions were pooled starting at 0.5 OD during the loading phase. After completion of loading, the column was washed with equilibration buffer (20 mM MES, 150 mM sodium acetate, pH 6.25) in a volume equal to 5 column volumes (CV) and all 5 CV were also collected.

The third and final step of hydrophobic interaction chromatography was run in breakthrough mode using PHENYL SEPHAROSE ^TM 6 Fast Flow (High Sub) resin. After equilibrating the column (50 mM sodium acetate pH 5.0), the pH of the anti-Abeta pool after the CAPTO ^™ Adhere column was adjusted to 5.0 and the pool was applied to the column. Anti-Abeta MAb passed through the column and the column was further washed with equilibration buffer (150 mM sodium acetate pH 5.0). A pool of anti-Abeta MAb was collected during the loading phase based on A280, starting to pool the 0.5 OD fractions. The column was washed with 10 CV of equilibration buffer (150 mM sodium acetate pH 5.0) and all 10 CV were also collected. As with the anti-IL13 MAb, additional virus inactivation and filtration steps and a final ultrafiltration-diafiltration (UFDF) step were included.

The results of applying the above process in four different purification experiments are shown in Table 8 below.

Table 8. PLBL2 levels at different stages of anti-Abeta MAb purification using HIC

Sample in the process of cleaning PLBL2
(ng/mg, according to ELISA) Experiment number 1 2 3 4 HCCF 622 564 1264 553 Pool after CpA 7 8 9 2.5 Pool after HIC (application density 300 g/l) 0.7 0.6 0.3 0.3 Pool after HIC (application density 10 g/l) < 0.2 < 0.2 < 0.2 Not tested

The results shown in Table 8 demonstrate that the use of the HIC resin at the end of the chromatography step effectively reduces the remaining levels of PLBL2 in the anti-Abeta MAb preparation to an amount comparable to that observed for the anti-IL13 MAb. The desired results in terms of product yield and reduction in PLBL2 levels were already obtained at 300 g/l loading density, however, as the loading density on the HIC column was reduced from 300 g/l to 100 g/l, a further decrease in the level of residual PLBL2 was observed.

We also investigated two other conditions for the HIC step, loading pH and loading molarity of sulfate. For these experiments, we started with a post-CAPTO ^™ Adhere pool containing 51 ng/mg PLBL2 (measured by ELISA), 15 mM sodium acetate, pH 5.5. We adjusted the application pH and the application sulfate molarity to the values shown in Table 9 below using stock solutions containing 0 mM sodium sulfate or 800 mM sodium sulfate at different pH values. We tested each application pH listed in Table 9 under low sulfate (0 mM) and high sulfate (240 mM) conditions. We tested each condition at an application density of 60 g/L. The results presented in Table 9 demonstrate that lowering the pH on application to pH 4 or pH 5, or increasing the molarity of sulfate on application (to 240 mM sulfate) was effective in reducing PLBL2 levels in the final post-HIC pool. The combination of pH 4.0 and 240 mM sulfate during application was particularly effective in minimizing the amount of PLBL2 remaining in the HIC pool.

Table 9. PLBL2 levels in pool after HIC for different application pH and application sulfate molarities

application pH PLBL2 (ng/mg, by ELISA) Low molarity sulfate
(0 mM) High molarity of sulfate
(240 mm) 4 4 1 5 10 3 6 27 5 7 64 6

Accordingly, the use of HIC resin at the end of the chromatography step in the purification of polypeptides produced in CHO cells, such as the anti-IL13 MAb and anti-Abeta MAb described herein, effectively reduces residual hamster PLBL2 to very low levels, for example, up to 1 ng/mg or less in the post-HIC pool.

Purification process to reduce hamster PLBL2 levels in IgG1 antibody preparations

We further assessed whether the purification methods described above for anti-IL13 and anti-Abeta IgG4 antibody preparations, in particular methods using a HIC column at the end of the chromatography, would also be effective in reducing PLBL2 levels in IgG1 antibody preparations. For these experiments, we chose an anti-IL17 A/F antibody, which is an IgG1 antibody, and is generated in CHO cells. Examples of anti-IL17 A/F antibodies and methods for producing such antibodies have been described previously, for example, in WO 2009136286 and US patent number 8,715,669. As described for anti-IL13 and anti-Abeta MAb, we tested various resins (particularly PHENYL SEPHAROSE ^TM FF [low sub] and PHENYL SEPHAROSE ^TM FF [high sub]) and buffers (particularly 50 mM sodium acetate, pH 5.5, and 50 mM Tris, 85 mM, sodium acetate, pH 8.0) for the HIC column to select those resins and buffers that are optimal in terms of quality and stability of the anti-IL17 A/F product, and in terms of removing PLBL2 hamster.

We found that a three-column process involving protein A affinity chromatography (MABSELECT SURE ^TM ), followed by cation exchange chromatography (POROS® 50HS) in binding and elution mode, and HIC (PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub)) in breakthrough collection mode was convenient and effective in reducing the level of PLBL2 in the final drug substance. This three-column process is detailed below.

The first affinity chromatography step was a coupling and elution process using MABSELECT SURE ^TM resin, similar to that described above for anti-IL13 MAb and anti-Abeta MAb.

The second stage of cation exchange chromatography was carried out in the mode of binding and elution using resin POROS® 50HS. After column equilibration (40 mM sodium acetate, pH 5.5), the pH of the anti-IL17 A/F pool after MABSELECT SURE ^TM was adjusted to pH 5.0 and the pool was applied to the column. The column was washed (40 mM sodium acetate, pH 5.5) and then the anti-IL17 A/F antibody was eluted from the column with a gradient of 40 to 400 mM sodium acetate, pH 5.5. Fractions were pooled based on A280 starting at ≥ 0.5 OD and ending at ≤ 2.0 OD during the gradient elution phase.

The third and final step of hydrophobic interaction chromatography was run in breakthrough mode using PHENYL SEPHAROSE ^TM 6 Fast Flow (High Sub) resin. After equilibrating the column (50 mM sodium acetate pH 5.5), the anti-IL17 A/F pool after the POROS® 50HS column was directly applied to the column without adjusting the pH. Anti-IL17 A/F MAb passed through the column. Anti-IL17 A/F MAb fractions were pooled based on A280 starting in the loading phase at ≥ 0.5 OD. The column was washed with 10 CV of equilibration buffer (50 mM sodium acetate, pH 5.5) and eluted fractions were collected during the wash phase and pooled at ≤ 1.0 OD.

The results of applying the above process during one purification experiment are shown in Table 10 below.

Table 10. PLBL2 levels at various stages of anti-IL17 A/F MAb purification using HIC

Sample in the process of cleaning PLBL2 (ng/mg, by ELISA) Experiment number 1 HCCF 713 Pool after MABSELECT SURE ^TM 151 Pool after POROS® 50HS 47 Pool after HIC (application density 100 g/l) < 0.7

The results shown in Table 10 demonstrate that the use of HIC resin at the end of the chromatography step effectively reduces the residual levels of PLBL2 in the anti-IL17 A/F MAb (IgG1) preparation to an amount comparable to that observed for anti-IL13 and anti -Abeta MAb (IgG4).

Anti-CMV Antibody

In addition to testing for anti-IL17 A/F, we tested another IgG1 MAb, an anti-CMV-MSL antibody, which was also generated in CHO cells. Examples of anti-CMV antibodies, including anti-CMV-MSL, and methods for making such antibodies have been described previously, for example, in WO 2012047732.

We again found that a three-column process involving protein A affinity chromatography (MABSELECT SURE ^TM ), followed by cation exchange chromatography (POROS® 50HS) in binding and elution mode, and HIC (PHENYL SEPHAROSE ^TM 6 Fast Flow (high sub) ) in breakthrough collection mode was convenient and effective in reducing the level of PLBL2 in the final drug substance. This three-column process is detailed below.

The first affinity chromatography step was a MABSELECT SURE ^™ resin binding and elution process similar to that described above for anti-IL13 MAb, anti-IL13, anti-Abeta and anti-IL17 A/F MAb.

The second stage of cation exchange chromatography was carried out in the mode of binding and elution using resin POROS® 50HS. After column equilibration (40 mM sodium acetate, pH 5.5), the pH of the anti-CMV-MSL pool after MABSELECT SURE ^TM was adjusted to pH 5.0 and the pool was applied to the column. The column was washed (40 mM sodium acetate, pH 5.5) and then the anti-CMV-MSL antibody was eluted from the column with a gradient of 40 to 400 mM sodium acetate, pH 5.5. Fractions were pooled based on A280 starting at ≥ 0.5 OD and ending at ≤ 1.0 OD during the gradient elution phase.

In this particular experiment, a viral filtration step was performed between the cation exchange chromatography and hydrophobic interaction chromatography steps using Viresolve Pro as a filter and a Fluorodyne UEDF filter as a pre-filter.

The third and final step of hydrophobic interaction chromatography was run in breakthrough mode using PHENYL SEPHAROSE ^TM 6 Fast Flow (High Sub) resin. After equilibrating the column (50 mM sodium acetate pH 5.5), the anti-CMV-MS pool after the POROS® 50HS column was directly applied to the column without adjusting the pH. Anti-CMV-MSMAb passed through the column. Anti-CMV-MS MAb fractions were pooled based on A280 starting in the loading phase at ≥ 0.5 OD. The column was washed with 10 CV of equilibrating buffer (50 mM sodium acetate, pH 5.5) and eluted fractions were collected during the wash phase and pooled at ≤ 0.5 OD.

The results of applying the above process during one purification experiment are shown in Table 11 below.

Table 11. PLBL2 levels at various stages of anti-CMV-MSL MAb purification with HIC

Sample in the process of cleaning PLBL2 (ng/mg, by ELISA) Experiment number 1 HCCF 2608 Pool after MABSELECT SURE ^TM 319 Pool after POROS® 50HS 33 Pool after Viresolve Pro 32 Pool after HIC (application density 60 g/l) < 0.6

The results shown in Table 11 demonstrate that the use of HIC resin in the final chromatography step resulted in an effective reduction of residual levels of PLBL2 in the anti-CMV-MSL MAb preparation to an amount comparable to that observed for anti-IL13, anti-Abeta and anti -IL17 A/F MAb. Accordingly, the use of HIC resin at the end of the chromatography step in the purification of polypeptides produced in CHO cells, such as the anti-IL13 MAbs and other MAbs described herein, effectively reduces the residual amount of hamster PLBL2 to very low levels, for example, to 1 ng/mg or less in the pool after HIC. Thus, we have shown that the use of the HIC step as described herein to reduce PLBL2 levels was as effective for MAb IgG1 as it was for MAb IgG4, demonstrating the applicability of this method, in general, to reduce levels Hamster PLBL2 in recombinant polypeptide preparations.

EXAMPLE 3 Evaluation of human antibody production against hamster PLBL2 in patients administered anti-IL13 MAb compositions containing varying amounts of hamster PLBL2

To evaluate the potential impact of PLBL2 CHO contamination, we developed an ELISA assay (an ELISA assay where the antibody acts as a bridge between immobilized and labeled antigens) to detect antibodies to hamster PLBL2 in human subjects who received the anti-IL13 MAb, lebrikizumab. Serum samples from patients participating in various clinical trials of lebrikizumab were analyzed for anti-hamster PLBL2 antibodies pre-dose and post-dose, and serum samples from placebo-treated patients were analyzed. Details of clinical studies have been described previously (WO 2012/083132, Corren et al., N Engl J Med 365:1088-98 (2011)), and only the most significant details of these studies are given below.

In an ELISA assay where antibody bridges between immobilized and labeled antigens, which was designed and validated to detect antibodies to hamster PLBL2 in human serum, two conjugated reagents were used to capture antibodies of any isotype against hamster PLBL2: purified hamster PLBL2 conjugated with biotin (biotin-PLBL2) and purified hamster PLBL2 conjugated to digoxigenin (DIG-PLBL2). Preparation and purification of hamster PLBL2 was performed using standard methods known to those skilled in the art and also described in U.S. Provisional Applications 61/877,503 and 61/991,228, and conjugation with biotin or DIG was performed using standard methods known to those skilled in the art. this field of technology. In this semi-homogeneous ELISA, where the antibody acts as a bridge between the immobilized and labeled antigens, 75 µl/well of a conjugation solution containing dilution solution (PBS/0.5% BSA/0.05% Polysorbate 20/0, 05% ProClin 300, pH 7.4 ± 0.1) and 3 µg/ml each of biotin-PLBL2 and DIG-PLBL2, were incubated in Micronic polypropylene tubes (National Scientific Supply Co.; Claremont, CA) overnight (16 - 24 hours) at ambient temperature together with 75 µl/well of 1:20 diluted serum samples and controls in dilution solution. After incubation, 100 µl/well mixtures were transferred from Micronic tubes to streptavidin-coated 96-well plates (StreptaWell™ High Bind; Roche Diagnostics; Indianapolis, IN) that had been previously washed 3 times with 400 µl/well wash buffer (PBS/0.05 % polysorbate 20) in an automatic plate washer (BioTek ELx405) and incubated at ambient temperature for 2 hours ± 10 minutes. The plate was washed 4 times with 400 µl/well of wash buffer in a plate washer, followed by the addition of 100 µl/well of mouse horseradish peroxidase (HRP)-conjugated anti-digoxin antibody (Jackson ImmunoResearch, catalog number 200-032-156), at a concentration of 400 ng/ml and incubated at ambient temperature for 2 hours ± 10 minutes for detection. After the plate was washed 4 times with 400 µl/well wash buffer in a plate washer, 100 µl/well of a mixture containing peroxidase substrate solution (tetramethylbenzidine) (0.4 g/l TMB) and peroxidase solution B was added to the wells of the plate. (0.02% hydrogen peroxide) (KPL, catalog number 50-76-03) at a ratio of 1:1, and incubated at ambient temperature for 18 - 28 minutes to develop color, after which the reaction was stopped by adding 100 μl / well with 1 M phosphoric acid. Absorbance was measured at 450 nm and at 630 nm for comparison. A monoclonal antibody construct containing a specific complementarity determining region (CDR) from a mouse anti-hamster PLBL2 antibody and a human IgG1 framework region was used as a positive control in the assay. The relative sensitivity of the assay using this antibody was 25 ng/mL. Drug tolerance assay experiments using this antibody demonstrated that serum levels of lebrikizumab up to 50 μg/mL or hamster PLBL2 concentrations up to 1 μg/mL did not result in confounding results or cross-reactivity in the assay.

To perform this assay, serum samples were first screened in this assay at a minimum dilution of 1/20. Samples that were screened to be seropositive were then used to confirm their specificity for hamster PLBL2 using a competitive confirmatory assay. If the sample was also determined to be seropositive, it was then serially diluted to obtain a titer value. Seropositive responses were recorded in titer units, which are the log10 dilution factor at which the signal from the sample is equal to the signal at the cutoff point of the assay (threshold for determining seropositivity).

Four clinical studies in which patient samples were analyzed using the anti-hamster PLBL2 antibody ELISA described above are briefly described below.

Study 1 was a Phase II, randomized, double-blind, placebo-controlled trial to validate the concept and evaluate the effects of lebrikizumab in patients with asthma who were not adequately controlled on long-term inhaled corticosteroid (ICS) therapy. A total of 219 patients were randomized, 106 of whom received at least one 250 mg subcutaneous (SC) dose of lebrikizumab and 92 received six monthly doses.

Study 2 was a Phase II randomized, double-blind, placebo-controlled study to determine the optimal dose in patients with asthma who were not treated with ICS. Patients received one of three doses (500, 250, or 125 mg) of lebrikizumab or placebo by SC administration. Study drug was administered four times over a period of 12 weeks. A total of 158 patients received at least one dose of lebrikizumab, and 145 patients received all four doses.

Study 3 was a Phase I study of the pharmacokinetics of lebrikizumab in healthy Japanese and Caucasian volunteers. Three separate cohorts of 20 healthy Japanese and Caucasian subjects (10 subjects of each racial group) were randomized to receive lebrikizumab (125, 250, and 375 mg SC) and placebo in a 7:3 ratio. Subjects received a single dose on day 1 and were followed up for 120 days thereafter. A total of 42 subjects received a single dose of lebrikizumab.

In studies 1-3, a total of 306 subjects took part, 264 of whom had asthma and received at least one dose of a substance containing hamster PLBL2. Hamster exposure to PLBL2 was different depending on the dose of lebrikizumab received.

Study 4 was a Phase IIb randomized, double-blind, placebo-controlled trial to evaluate the efficacy and safety of lebrikizumab in patients with uncontrolled asthma who used ICS and a second control agent. Patients received one of three doses (250, 125, or 37.5 mg) of lebrikizumab or placebo via SC administration monthly. A total of 463 patients were randomized in Study 4, 347 of whom received at least one dose of lebrikizumab. Hamster exposure to PLBL2 was different depending on the dose of lebrikizumab received.

Table 12 below summarizes the information for each Study 1-4, reflecting the range of hamster PLBL2 levels in exposed subjects and doses of lebrikizumab.

Table 12 Hamster PLBL2 Exposure in Lebrikizumab Clinical Trials

Study PLBL2 in drug substance (ng/mg) Lebrikizumab dose (mg/month) PLBL2 (mcg/dose) 1 ^34-137a 250 9-34 2 ^34-137a 125 4-17 250 9-34 500 17-69 3 34 125 4 250 9 375 13 4 242 37.5 9 328 125 41 328 250 82

^a Range across four different clinical product lots.

A retrospective analysis of selected time points from Study 1 was performed using the anti-hamster PLBL2 antibody assay described above to detect anti-hamster PLBL2 antibodies. Both samples from placebo-treated subjects and samples from lebrikizumab-treated subjects were analyzed to determine the level of pre-existing response as well as to assess antibody production in response to lebrikizumab. There were 113 subjects in the placebo group and 106 subjects received at least one dose of lebrikizumab. The following time points were chosen for the analysis: 0, 29, 85, 141, 225 days and early completion. Samples were taken before the next dose; thus, samples obtained on day 29 were taken prior to the second dose. The percentage of subjects seropositive for anti-hamster PLBL2 antibodies at each time point was calculated as the ratio of the number of seropositive subjects at each time point to the total number of subjects tested at each time point. Data are shown in Table 13.

Table 13. Results for anti-hamster PLBL2 antibody in Study 1

% of seropositive subjects at each time point (number of seropositive subjects/total number of subjects to be evaluated) Research Day 0 29 85 141 225 Early termination placebo 6 (7/110) 7 (8/107) 9 (9/104) 8 (8/99) 5 (5/97) 25 (2/8) Dose 250 mg 5 (5/102) 6 (6/100) 89 (90/101) 98 (92/94) 98 (91/93) 100 (8/8) ^a

^a Of the 8 subjects who received lebrikizumab and terminated the drug study early, only 3 experienced adverse events leading to the termination of the drug study.

The 6 placebo subjects in Study 1 were seropositive prior to dosing on day 0 and remained seropositive throughout the study. That samples from these subjects are seropositive was confirmed by a confirmatory competitive assay, with antibody titers at day 0 ranging from 1.6 to 2.9 titer units. Antibody titers obtained at subsequent visits were the same as those obtained on day 0. Several other placebo subjects also experienced a low-level positive response during the Study.

Among subjects treated with lebrikizumab in Study 1, 98% (104/106) were antibody seropositive (responding positive) after dosing and remained seropositive until the end of the study, with the majority of subjects becoming seropositive after receiving at least two doses of lebrikizumab. Post-dose titers ranged from 1.35 to 4.76 titer units, with titers typically rising over time. The clinical significance of antibody production against hamster PLBL2 is unknown. No clinically significant hazards were identified in this study, and given the high prevalence of antibodies to hamster PLBL2, it was not possible to establish a correlation between antibody production and safety events.

An interim analysis was also performed on the samples collected in Study 4. Both samples from subjects receiving placebo and samples from subjects receiving treated with a dose of lebrikizumab. In total, there were 116 subjects who received placebo and 347 subjects who received at least one dose of lebrikizumab. Samples from 92 placebo-treated subjects and 268 lebrikizumab-treated subjects are presented in this dataset. The results are shown in Table 14.

Table 14 Results for anti-hamster PLBL2 antibody in Study 4 for subjects not previously exposed to Lebrikizumab.

% of seropositive subjects at each time point (number of seropositive subjects/total number of subjects to be evaluated) Research Day 0 29 85 169 253 Early termination placebo 4 (4/89) 4 (3/78) 4 (2/48) 0 (0/13) NA 0 (0/5) Dose 37.5 mg 9 (8/88) 9 (7/82) 55 (35/64) 79 (27/34) 66 (2/3) 43 (3/7) ^a Dose 125 mg 4 (3/81) 11 (8/73) 87 (48/55) 100 (9/9) NA 0 (0/2) ^a Dose 250 mg 5 (4/88) 10 (7/72) 96 (49/51) 100 (13/13) NA 67 (2/3) ^a

^a Of the 12 subjects who received lebrikizumab and terminated the drug study early, only 4 experienced adverse events leading to the termination of the drug study.

The four placebo-treated subjects in Study 4 who were seropositive prior to dosing on day 0 had a low-level positive response that was slightly above the limit of detection in the assay. Low-level responses were also observed at some but not all subsequent time points.

The 15 lebrikizumab-treated subjects in Study 4 who were seropositive prior to dosing on day 0 remained seropositive at subsequent time points, with antibody titer increasing after multiple doses. In addition, Study 4 included 10 subjects who had previously received lebrikizumab in Study 1. Nine of these subjects were further re-treated with lebrikizumab in Study 4, and 1 subject received placebo. All 10 subjects were seropositive prior to dosing on Day 0 of Study 4 and continued to be seropositive at subsequent time points. Data for these 10 subjects were excluded from Table 14 due to their previous exposure to lebrikizumab.

Among subjects treated with lebrikizumab in Study 4, differences in positive response rates were observed between groups receiving different doses. However, since these data are incomplete, conclusions about the significance of these differences cannot be drawn at this time. Similar to Study 1, most subjects became seropositive after receiving at least two doses of lebrikizumab. Post-dose titers ranged from 1.68 to 4.55 titer units, with titers typically rising over time. Since this data set is incomplete, the percentage of seropositive subjects and the range of titer values may change as more data is accumulated.

The Interim Safety Assessment in Study 4 resulted in a profile similar to that obtained in early completed studies in which no clinically significant adverse events were reported, including anaphylaxis or severe hypersensitivity reactions. Of note, 6 of 9 patients treated with lebrikizumab in Study 1 and then re-treated with lebrikizumab in Study 4 had no side effects during the pre-test, and only 1 patient experienced local reactions at the injection site. To date, no clinical implications of this anti-hamster PLBL2 antibody response have been identified in clinical trials.

We also evaluated in the 125 mg dose group in Study 2 and these results are shown in Table 15.

Table 15 Results for anti-hamster PLBL2 antibody in Study 2

% of seropositive subjects at each time point (number of seropositive subjects/total number of subjects to be evaluated) Research Day 0 29 57 85 141 Early termination Dose 125 mg 4 (2/51) 21 (11/53) 70 (35/50) 88 (45/51) 86 (43/50) 100 (2/2) ^a

^a Of the 2 subjects who received lebrikizumab and terminated the drug study early, none experienced side effects that could lead to the termination of the drug study.

The two subjects in Study 2 who were seropositive prior to dosing on day 0 continued to be seropositive at all subsequent time points, with the titer increasing after multiple doses. Among subjects in Study 2 treated with 125 mg lebrikizumab, 87% (46/53) were antibody seropositive post-dose and remained seropositive until the end of the study, with the majority of subjects becoming seropositive after receiving at least two doses of lebrikizumab. Post-dose titers ranged from 1.51 to 4.09 titer units, with titers typically rising over time.

Conclusions

To evaluate the potential impact of PLBL2 CHO contamination, we designed an assay to detect antibodies to hamster PLBL2 in subjects treated with lebrikizumab preparations containing significant levels of hamster PLBL2. Based on the full data set from Study 1 and Study 2 for the 125 mg dose group, and also based on the partial data set from Study 4, it was concluded that the presence of hamster PLBL2 in lebrikizumab formulations elicited an immune response in the majority of subjects exposed to exposed to PLBL2 hamster.

A number of subjects in both the placebo group and the different doses of lebrikizumab groups exhibited pre-existing immunoreactivity in the anti-hamster PLBL2 antibody assay. The reason for such a pre-existing response is unknown; antibody reactivity to CHO host cell proteins has been previously described and validated in normal human serum samples not previously exposed to biological products derived from CHO (Xue et al., The AAPS Journal 12(1):98-106 (2010) ). However, there are no published data on a single species CHOP, PLBL2.

In subjects with pre-existing immunoreactivity, as determined by the anti-hamster PLBL2 antibody assay at baseline, a sustained increase in antibody titers was observed following lebrikizumab administration. In the case of subjects who did not have such antibodies at baseline, the majority of subjects in all four studies became seropositive after at least two administrations of lebrikizumab and remained seropositive at all time points.

The clinical relevance of antibody production against hamster PLBL2 is not known. Although the study subjects had high levels of antibodies to hamster PLBL2, it was not possible to establish a correlation between the presence of antibodies and safety-related events. Importantly, no hazards were identified in these completed or interim studies and, in particular, there were no reported cases of anaphylaxis, anaphylactoid reactions, or severe hypersensitivity reactions. However, there remains concern that prolonged exposure to repeated doses may increase the likelihood of adverse effects such as anaphylaxis, hypersensitivity, and immune complex deposition, especially in the asthma patient population and in the patient population with other allergic or hypersensitivity reactions. Accordingly, at the late stage of clinical trials and beyond, when repeated doses of anti-IL13 MAbs may be used over an extended period of time, it is important for patients to administer anti-IL13 MAbs (eg, lebrikizumab) that contain significantly reduced levels of hamster PLBL2 to minimize immunogenicity. as much as possible.

Additional antibody sequences are provided in Table 16 below.

Table 16. Amino acid sequences of anti-IL17 A/F antibody (SEQ ID NOS.: 15-22) and amino acid sequences of anti-Abeta antibody (SEQ ID NOS.: 23-30).

CDR-H1
(SEQ ID NO.:15) Asp Tyr Ala Met His CDR-H2
(SEQ ID NO.:16) Gly Ile Asn Trp Ser Ser Gly Gly Ile Gly Tyr Ala Asp Ser Val Lys Gly CDR-H3
(SEQ ID NO.:17) Asp Ile Gly Gly Phe Gly Glu Phe Tyr Trp Asn Phe Gly Leu CDR-L1
(SEQ ID NO.:18) Arg Ala Ser Gln Ser Val Arg Ser Tyr Leu Ala CDR-L2
(SEQ ID NO.:19) Asp Ala Ser Asn Arg Ala Thr CDR-L3
(SEQ ID NO.:20) Gln Gln Arg Ser Asn Trp Pro Pro Ala Thr vh
(SEQ ID NO.:21) Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val
Ser Gly Ile Asn Trp Ser Ser Gly Gly Ile Gly Tyr Ala Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
Ala Arg Asp Ile Gly Gly Phe Gly Glu Phe Tyr Trp Asn Phe Gly Leu
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser VL
(SEQ ID NO.:22) Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
Ala Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys CDR-H1
(SEQ ID NO.:23) GFTFSSYGMS CDR-H2
SEQ ID NO.:24) SINSNGGSTY YPDSVK CDR-H3
SEQ ID NO.: 25) GDY CDR-L1
(SEQ ID NO.:26) RSSQSLVYSN GDTYLH CDR-L2
(SEQ ID NO.: 27) KVSNRFS CDR-L3
(SEQ ID NO.: 28) SQSTHVPWT vh
(SEQ ID NO.: 29) EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYGMSWVRQA PGKGLELVAS INSNGGSTYY PDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCASGD YWGQGTTVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT KTYTCNVDHK PSNTKVDKRV ESKYGPPCPP CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLG VL
(SEQ ID NO.: 30) DIVMTQSPLS LPVTPGEPAS ISCRSSQSLV YSNGDTYLHW YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCSQSTHVP WTFGQGTKVE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

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polypeptide

<400> 12

Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr

1 5 10 15

Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr Ser

20 25 30

Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala

35 40 45

Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys Ser

50 55 60

Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr

65 70 75 80

Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Gly

85 90 95

Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 13

<211> 445

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<400> 13

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ala Tyr

20 25 30

Ser Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Ala Met Ile Trp Gly Asp Gly Lys Ile Val Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu

65 70 75 80

Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Gly Asp Gly Tyr Tyr Pro Tyr Ala Met Asp Asn Trp Gly Gln Gly Ser

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 14

<211> 218

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<400> 14

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ser Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Lys Ser Val Asp Ser Tyr

20 25 30

Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn

85 90 95

Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 15

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 15

Asp Tyr Ala Met His

15

<210> 16

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 16

Gly Ile Asn Trp Ser Ser Gly Gly Ile Gly Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 17

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 17

Asp Ile Gly Gly Phe Gly Glu Phe Tyr Trp Asn Phe Gly Leu

1 5 10

<210> 18

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 18

Arg Ala Ser Gln Ser Val Arg Ser Tyr Leu Ala

1 5 10

<210> 19

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 19

Asp Ala Ser Asn Arg Ala Thr

15

<210> 20

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 20

Gln Gln Arg Ser Asn Trp Pro Pro Ala Thr

1 5 10

<210> 21

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<400> 21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ser Gly Ile Asn Trp Ser Ser Gly Gly Ile Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Asp Ile Gly Gly Phe Gly Glu Phe Tyr Trp Asn Phe Gly Leu

100 105 110

Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<400> 22

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ala Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 23

Gly Phe Thr Phe Ser Tyr Gly Met Ser

1 5 10

<210> 24

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 24

Ser Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val Lys

1 5 10 15

<210> 25

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 25

Gly Asp Tyr

1

<210> 26

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 26

Arg Ser Ser Gln Ser Leu Val Tyr Ser Asn Gly Asp Thr Tyr Leu His

1 5 10 15

<210> 27

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 27

Lys Val Ser Asn Arg Phe Ser

15

<210> 28

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 28

Ser Gln Ser Thr His Val Pro Trp Thr

15

<210> 29

<211> 438

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val

35 40 45

Ala Ser Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

100 105 110

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

115 120 125

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

130 135 140

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

145 150 155 160

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

165 170 175

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

180 185 190

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

195 200 205

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

305 310 315 320

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Ser Gln Glu Glu Met Thr Lys

340 345 350

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser Leu Gly

435

<210> 30

<211> 219

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

polypeptide

<400> 30

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser

20 25 30

Asn Gly Asp Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 31

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 31

Ser Val Leu Leu Asp Ala Ala Ser Gly Gln Leu Arg

1 5 10

<210> 32

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 32

Gly Leu Glu Asp Ser Tyr Glu Gly Arg

15

<210> 33

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 33

Ala Phe Ile Pro Asn Gly Pro Ser Pro Gly Ser Arg

1 5 10

<210> 34

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic

peptide

<400> 34

Val Thr Ser Phe Ser Leu Ala Lys

15

<---

Claims (35)

1. A method for purifying a recombinant polypeptide produced in Chinese hamster ovary host cells having phospholipase B-like enzyme 2 (PLBL2) levels greater than 100 ng/mg, comprising a hydrophobic interaction chromatography (HIC) step, wherein the method provides a purified preparation, comprising a recombinant polypeptide and a residual amount of hamster PLBL2. 2. The method of claim 1, wherein the residual amount of hamster PLBL2 is less than 20 ng/mg, or less than 15 ng/mg, or less than 10 ng/mg, or less than 8 ng/mg, or less than 5 ng/mg, or less 3 ng/mg, or less than 2 ng/mg, or less than 1 ng/mg, or less than 0.5 ng/mg. 3. The method of claim 1 or 2 wherein the HIC step comprises the use of a PHENYL SEPHAROSE™ 6 Fast Flow high replacement resin. 4. The method of claim 3 wherein the HIC step comprises using a PHENYL SEPHAROSE™ 6 Fast Flow high replacement resin column in breakthrough mode. 5. The method according to any one of paragraphs. 1-4, wherein the recombinant polypeptide is selected from a growth factor, a cytokine, an antibody, an antibody fragment, and an immunoadhesin. 6. The method of claim 5, wherein the recombinant polypeptide is an antibody. 7. The method of claim 6 wherein the antibody is a humanized monoclonal antibody. 8. The method of claim 7 wherein the antibody is IgG1 or IgG2 or IgG3 or IgG4. 9. The method of claim 1 or 2, wherein the HIC step comprises using a resin column in breakthrough collection mode and includes using an equilibration buffer and a wash buffer, where the equilibration buffer and wash buffer each comprise 50 mM sodium acetate, pH 5.0. 10. The method of claim 9, wherein the breakthrough is monitored by absorbance at 280 nanometers and the breakthrough is collected between 0.5 and 1.5 optical density (OD). 11. The method of claim 9 wherein the breakthrough is collected in a maximum of 8 column volumes. 12. The method of claim 9, further comprising an affinity chromatography step. 13. The method of claim 12, wherein the affinity chromatography is Protein A chromatography. 14. The method of claim 9, further comprising the step of ion exchange chromatography. 15. The method of claim 14 wherein the ion exchange chromatography is an anion exchange chromatography. 16. The method of claim 9, comprising a first Protein A affinity chromatography step and a second anion exchange chromatography step prior to a hydrophobic interaction chromatography (HIC) step. 17. The method of claim 16 wherein the affinity chromatography step comprises MABSELECT SURE™ resin, the anion exchange chromatography step comprises Q SEPHAROSE™ Fast Flow resin, and the HIC step comprises PHENYL SEPHAROSE™ 6 Fast Flow high substitution resin. 18. The method according to claim 17, where: the affinity chromatography step includes the use of a MABSELECT SURE™ resin column in binding and elution mode; the anion exchange chromatography step includes using a Q SEPHAROSE™ Fast Flow Resin Column in Binding and Elution mode, and the HIC step involves using a PHENYL SEPHAROSE™ 6 Fast Flow high replacement resin column in breakthrough mode. 19. The method of claim 1 or 2, wherein the HIC step comprises using a resin column in breakthrough collection mode and includes using an equilibration buffer and wash buffer, where the equilibration buffer and wash buffer each comprise 150 mM sodium acetate, pH 5.0. 20. The method of claim 19 wherein the breakthrough is monitored by absorbance at 280 nanometers and the breakthrough is collected starting at 0.5 optical density (OD) and in a volume of 10 column volumes. 21. The method of claim 19, further comprising an affinity chromatography step. 22. The method of claim 21 wherein the affinity chromatography is protein A chromatography. 23. The method of claim 19, further comprising a combined chromatography step. 24. The method of claim 19, comprising a first Protein A affinity chromatography step and a second combined chromatography step prior to a hydrophobic interaction chromatography (HIC) step. 25. The method of claim 24, wherein the affinity chromatography step comprises the use of MABSELECT SURE™ resin, the combined chromatography step comprises the use of CAPTO™ Adhere, and the HIC step comprises the use of PHENYL SEPHAROSE™ 6 Fast Flow high substitution resin. 26. The method according to claim 25, where: the affinity chromatography step includes the use of a MABSELECT SURE™ resin column in binding and elution mode; the combined chromatography step includes using a CAPTO™ Adhere resin column in breakthrough mode and the HIC step involves using a PHENYL SEPHAROSE™ 6 Fast Flow high replacement resin column in breakthrough mode. 27. The method of claim 26, wherein the amount of hamster PLBL2 is quantified using immunoassay or mass spectrometric analysis. 28. The method of claim 27, wherein the immunoassay is an ELISA for all proteins of Chinese hamster ovary cells or an ELISA for hamster PLBL2. 29. The method of claim 27 wherein the mass spectrometry analysis is LC-MS/MS.

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